SPARCLE, a p53-induced lncRNA, controls apoptosis after genotoxic stress by promoting PARP-1 cleavage.

p53, master transcriptional regulator of the genotoxic stress response, controls cell-cycle arrest and apoptosis following DNA damage. Here, we identify a p53-induced lncRNA suicidal PARP-1 cleavage enhancer (SPARCLE) adjacent to miR-34b/c required for p53-mediated apoptosis. SPARCLE is a ∼770-nt, nuclear lncRNA induced 1 day after DNA damage. Despite low expression (<16 copies/cell), SPARCLE deletion increases DNA repair and reduces DNA-damage-induced apoptosis as much as p53 deficiency, while its overexpression restores apoptosis in p53-deficient cells. SPARCLE does not alter gene expression. SPARCLE binds to PARP-1 with nanomolar affinity and causes apoptosis by acting as a caspase-3 cofactor for PARP-1 cleavage, which separates PARP-1's N-terminal (NT) DNA-binding domain from its catalytic domains. NT-PARP-1 inhibits DNA repair. Expressing NT-PARP-1 in SPARCLE-deficient cells increases unrepaired DNA damage and restores apoptosis after DNA damage. Thus, SPARCLE enhances p53-induced apoptosis by promoting PARP-1 cleavage, which interferes with DNA-damage repair.

P-TEFb Activation by RBM7 Shapes a Pro-survival Transcriptional Response to Genotoxic Stress.

DNA damage response (DDR) involves dramatic transcriptional alterations, the mechanisms of which remain ill defined. Here, we show that following genotoxic stress, the RNA-binding motif protein 7 (RBM7) stimulates RNA polymerase II (Pol II) transcription and promotes cell viability by activating the positive transcription elongation factor b (P-TEFb) via its release from the inhibitory 7SK small nuclear ribonucleoprotein (7SK snRNP). This is mediated by activation of p38MAPK, which triggers enhanced binding of RBM7 with core subunits of 7SK snRNP. In turn, P-TEFb relocates to chromatin to induce transcription of short units, including key DDR genes and multiple classes of non-coding RNAs. Critically, interfering with the axis of RBM7 and P-TEFb provokes cellular hypersensitivity to DNA-damage-inducing agents due to activation of apoptosis. Our work uncovers the importance of stress-dependent stimulation of Pol II pause release, which enables a pro-survival transcriptional response that is crucial for cell fate upon genotoxic insult.

Genotoxic stress impacts pre-mRNA 3'-end processing.

Genotoxic stress, arising from various environmental sources and endogenous cellular processes, pose a constant threat to genomic stability. Cells have evolved intricate mechanisms to detect and repair DNA damage, orchestrating a robust genotoxic stress response to safeguard the integrity of the genome. Recent research has shed light on the crucial role of co- and post-transcriptional regulatory mechanisms in modulating the cellular response to genotoxic stress. Here we highlight recent advances illustrating the intricate interplay between pre-mRNA processing, with a focus on 3'-end processing, and genotoxic stress response.

Dissecting Ubiquitylation and DNA Damage Response Pathways in the Yeast Saccharomyces cerevisiae Using a Proteome-Wide Approach.

In response to genotoxic stress, cells evolved with a complex signaling network referred to as the DNA damage response (DDR). It is now well established that the DDR depends upon various posttranslational modifications; among them, ubiquitylation plays a key regulatory role. Here, we profiled ubiquitylation in response to the DNA alkylating agent methyl methanesulfonate (MMS) in the budding yeast Saccharomyces cerevisiae using quantitative proteomics. To discover new proteins ubiquitylated upon DNA replication stress, we used stable isotope labeling by amino acids in cell culture, followed by an enrichment of ubiquitylated peptides and LC-MS/MS. In total, we identified 1853 ubiquitylated proteins, including 473 proteins that appeared upregulated more than 2-fold in response to MMS treatment. This enabled us to localize 519 ubiquitylation sites potentially regulated upon MMS in 435 proteins. We demonstrated that the overexpression of some of these proteins renders the cells sensitive to MMS. We also assayed the abundance change upon MMS treatment of a selection of yeast nuclear proteins. Several of them were differentially regulated upon MMS treatment. These findings corroborate the important role of ubiquitin-proteasome-mediated degradation in regulating the DDR.

What is the role of current mass spectrometry in pharmaceutical analysis?

The role of mass spectrometry (MS) has become more important in most application domains in recent years. Pharmaceutical analysis is specific due to its stringent regulation procedures, the need for good laboratory/manufacturing practices, and a large number of routine quality control analyses to be carried out. The role of MS is, therefore, very different throughout the whole drug development cycle. While it dominates within the drug discovery and development phase, in routine quality control, the role of MS is minor and indispensable only for selected applications. Moreover, its role is very different in the case of analysis of small molecule pharmaceuticals and biopharmaceuticals. Our review explains the role of current MS in the analysis of both small-molecule chemical drugs and biopharmaceuticals. Important features of MS-based technologies being implemented, method requirements, and related challenges are discussed. The differences in analytical procedures for small molecule pharmaceuticals and biopharmaceuticals are pointed out. While a single method or a small set of methods is usually sufficient for quality control in the case of small molecule pharmaceuticals and MS is often not indispensable, a large panel of methods including extensive use of MS must be used for quality control of biopharmaceuticals. Finally, expected development and future trends are outlined.

The DNA damage sensor ATM kinase interacts with the p53 mRNA and guides the DNA damage response pathway.

BACKGROUND: The ATM kinase constitutes a master regulatory hub of DNA damage and activates the p53 response pathway by phosphorylating the MDM2 protein, which develops an affinity for the p53 mRNA secondary structure. Disruption of this interaction prevents the activation of the nascent p53. The link of the MDM2 protein-p53 mRNA interaction with the upstream DNA damage sensor ATM kinase and the role of the p53 mRNA in the DNA damage sensing mechanism, are still highly anticipated. METHODS: The proximity ligation assay (PLA) has been extensively used to reveal the sub-cellular localisation of the protein-mRNA and protein-protein interactions. ELISA and co-immunoprecipitation confirmed the interactions in vitro and in cells. RESULTS: This study provides a novel mechanism whereby the p53 mRNA interacts with the ATM kinase enzyme and shows that the L22L synonymous mutant, known to alter the secondary structure of the p53 mRNA, prevents the interaction. The relevant mechanistic roles in the DNA Damage Sensing pathway, which is linked to downstream DNA damage response, are explored. Following DNA damage (double-stranded DNA breaks activating ATM), activated MDMX protein competes the ATM-p53 mRNA interaction and prevents the association of the p53 mRNA with NBS1 (MRN complex). These data also reveal the binding domains and the phosphorylation events on ATM that regulate the interaction and the trafficking of the complex to the cytoplasm. CONCLUSION: The presented model shows a novel interaction of ATM with the p53 mRNA and describes the link between DNA Damage Sensing with the downstream p53 activation pathways; supporting the rising functional implications of synonymous mutations altering secondary mRNA structures.

Xanthine oxidoreductase mediates genotoxic drug-induced autophagy and apoptosis resistance by uric acid accumulation and TGF-ß-activated kinase 1 (TAK1) activation.

Autophagy is a highly conserved cellular process that profoundly impacts the efficacy of genotoxic chemotherapeutic drugs. TGF-ß-activated kinase 1 (TAK1) is a serine/threonine kinase that activates several signaling pathways involved in inducing autophagy and suppressing cell death. Xanthine oxidoreductase (XOR) is a rate-limiting enzyme that converts hypoxanthine to xanthine, and xanthine to uric acid and hydrogen peroxide in the purine catabolism pathway. Recent studies showed that uric acid can bind to TAK1 and prolong its activation. We hypothesized that genotoxic drugs may induce autophagy and apoptosis resistance by activating TAK1 through XOR-generated uric acid. Here, we report that gemcitabine and 5-fluorouracil (5-FU), two genotoxic drugs, induced autophagy in HeLa and HT-29 cells by activating TAK1 and its two downstream kinases, AMP-activated kinase (AMPK) and c-Jun terminal kinase (JNK). XOR knockdown and the XOR inhibitor allopurinol blocked gemcitabine-induced TAK1, JNK, AMPK, and Unc51-like kinase 1 (ULK1)S555 phosphorylation and gemcitabine-induced autophagy. Inhibition of the ATM-Chk pathway, which inhibits genotoxic drug-induced uric acid production, blocked gemcitabine-induced autophagy by inhibiting TAK1 activation. Exogenous uric acid in its salt form, monosodium urate (MSU), induced autophagy by activating TAK1 and its downstream kinases JNK and AMPK. Gene knockdown or the inhibitors of these kinases blocked gemcitabine- and MSU-induced autophagy. Inhibition of autophagy by allopurinol, chloroquine, and 5Z-7-oxozeaenol (5Z), a TAK1-specific inhibitor, enhanced gemcitabine-induced apoptosis. Our study uncovers a previously unrecognized role of XOR in regulating genotoxic drug-induced autophagy and apoptosis and has implications for designing novel therapeutic strategies for cancer treatment.

Styrene lung cancer risk assessment: an alternative evaluation of human lung cancer risk assuming mouse lung tumors are potentially human relevant and operating by a threshold-based non-genotoxic mode of action.

Rodent inhalation studies indicate styrene is a mouse lung-specific carcinogen. Mode-of-action (MOA) analyses indicate that the lung tumors cannot be excluded as weakly quantitatively relevant to humans due to shared oxidative metabolites detected in rodents and humans. However, styrene also is not genotoxic following in vivo dosing. The objective of this review was to characterize occupational and general population cancer risks by conservatively assuming mouse lung tumors were relevant to humans but operating by a non-genotoxic MOA. Inhalation cancer values reference concentrations for respective occupational and general population exposures (RfCcar-occup and RfCcar-genpop) were derived from initial benchmark dose (BMD) modeling of mouse inhalation tumor dose-response data. An overall lowest BMDL10 of 4.7 ppm was modeled for lung tumors, which was further duration- and dose-adjusted by physiologically based pharmacokinetic (PBPK) modeling to derive RfCcar-occup/genpop values of 6.2 ppm and 0.8 ppm, respectively. With the exception of open-mold fiber reinforced composite workers not using personal protective equipment (PPE), the RfCcar-occup/genpop values are greater than typical occupational and general population human exposures, thus indicating styrene exposures represent a low potential for human lung cancer risk. Consistent with this conclusion, a review of styrene occupational epidemiology did not support a conclusion of an association between styrene exposure and lung cancer occurrence, and further supports a conclusion that the conservatively derived RfCcar-occup is lung cancer protective.

Corelease of Genotoxic Polycyclic Aromatic Hydrocarbons and Nanoparticles from a Commercial Aircraft Jet Engine - Dependence on Fuel and Thrust.

Jet engines are important contributors to global CO2 emissions and release enormous numbers of ultrafine particles into different layers of the atmosphere. As a result, aviation emissions are affecting atmospheric chemistry and promote contrail and cloud formation with impacts on earth's radiative balance and climate. Furthermore, the corelease of nanoparticles together with carcinogenic polycyclic aromatic hydrocarbons (PAHs) affects air quality at airports. We studied exhausts of a widely used turbofan engine (CFM56-7B26) operated at five static thrust levels (idle, 7, 30, 65, and 85%) with conventional Jet A-1 fuel and a biofuel blend composed of hydro-processed esters and fatty acids (HEFA). The particles released, the chemical composition of condensable material, and the genotoxic potential of these exhausts were studied. At ground operation, particle number emissions of 3.5 and 0.5 × 1014 particles/kg fuel were observed with highest genotoxic potentials of 41300 and 8800 ng toxicity equivalents (TEQ)/kg fuel at idle and 7% thrust, respectively. Blending jet fuel with HEFA lowered PAH and particle emissions by 7-34% and 65-67% at idle and 7% thrust, respectively, indicating that the use of paraffin-rich biofuels is an effective measure to reduce the exposure of airport personnel to nanoparticles coated with genotoxic PAHs (Trojan horse effect).

Cyclin F-Mediated Degradation of SLBP Limits H2A.X Accumulation and Apoptosis upon Genotoxic Stress in G2.

SLBP (stem-loop binding protein) is a highly conserved factor necessary for the processing, translation, and degradation of H2AFX and canonical histone mRNAs. We identified the F-box protein cyclin F, a substrate recognition subunit of an SCF (Skp1-Cul1-F-box protein) complex, as the G2 ubiquitin ligase for SLBP. SLBP interacts with cyclin F via an atypical CY motif, and mutation of this motif prevents SLBP degradation in G2. Expression of an SLBP stable mutant results in increased loading of H2AFX mRNA onto polyribosomes, resulting in increased expression of H2A.X (encoded by H2AFX). Upon genotoxic stress in G2, high levels of H2A.X lead to persistent γH2A.X signaling, high levels of H2A.X phosphorylated on Tyr142, high levels of p53, and induction of apoptosis. We propose that cyclin F co-evolved with the appearance of stem-loops in vertebrate H2AFX mRNA to mediate SLBP degradation, thereby limiting H2A.X synthesis and cell death upon genotoxic stress.

TRAF6 Restricts p53 Mitochondrial Translocation, Apoptosis, and Tumor Suppression.

Mitochondrial p53 is involved in apoptosis and tumor suppression. However, its regulation is not well studied. Here, we show that TRAF6 E3 ligase is a crucial factor to restrict mitochondrial translocation of p53 and spontaneous apoptosis by promoting K63-linked ubiquitination of p53 at K24 in cytosol, and such ubiquitination limits the interaction between p53 and MCL-1/BAK. Genotoxic stress reduces this ubiquitination in cytosol by S13/T330 phosphorylation-dependent translocation of TRAF6 from cytosol to nucleus, where TRAF6 also facilitates the K63-linked ubiquitination of nuclear p53 and its transactivation by recruiting p300 for p53 acetylation. Functionally, K63-linked ubiquitination of p53 compromised p53-mediated apoptosis and tumor suppression. Colorectal cancer samples with WT p53 reveal that TRAF6 overexpression negatively correlates with apoptosis and predicts poor response to chemotherapy and radiotherapy. Together, our study identifies TRAF6 as a critical gatekeeper to restrict p53 mitochondrial translocation, and such mechanism may contribute to tumor development and drug resistance.

Escaping the cohort of concern: in vitro experimental evidence supports non-mutagenicity of N-nitroso-hydrochlorothiazide.

In recent years, nitrosamine impurities in pharmaceuticals have been subject to intense regulatory scrutiny, with nitrosamine drug substance-related impurities (NDSRIs) treated as cohort of concern impurities, regardless of predicted mutagenic potential. Here, we describe a case study of the NDSRI N-nitroso-hydrochlorothiazide (NO-HCTZ), which was positive in the bacterial reverse mutation (Ames) test but is unstable under the test conditions, generating formaldehyde among other products. The mutagenic profile of NO-HCTZ was inconsistent with that expected of a mutagenic nitrosamine, exhibiting mutagenicity in the absence of metabolic activation, and instead aligned well with that of formaldehyde. To assess further, a modified Ames system including glutathione (3.3 mg/plate) to remove formaldehyde was developed. Strains used were S. typhimurium TA98, TA100, TA1535, and TA1537, and E. coli WP2 uvrA/pKM101. In this system, formaldehyde levels were considerably lower, with a concomitant increase in levels of S-(hydroxymethyl)glutathione (the adduct formed between glutathione and formaldehyde). Upon retesting NO-HCTZ in the modified system (1.6-5000 µg/plate), a clear decrease in the mutagenic response was observed in the strains in which NO-HCTZ was mutagenic in the original system (TA98, TA100, and WP2 uvrA/pKM101), indicating that formaldehyde drives the response, not NO-HCTZ. In strain TA1535, an increase in revertant colonies was observed in the modified system, likely due to a thiatriazine degradation product formed from NO-HCTZ under Ames test conditions. Overall, these data support a non-mutagenic designation for NO-HCTZ and demonstrate the value of further investigation when a positive Ames result does not align with the expected profile.

A modular strategy for the testing and assessment of non-genotoxic carcinogens.

A modular strategy is described for the testing and assessment (MoSt) of non-genotoxic carcinogenicity (NGTxC) that is suitable for regulatory applications. It utilizes and builds upon work conducted by the OECD expert group on NGTxC. The approach integrates relevant test methods from the molecular- to cellular- and further to tissue level, many of which have been recently reviewed. Six progressive modules are included in the strategy. Advice is provided for the iterative selection of the next appropriate test method within each step of the strategy. Assessment is completed by a weight of evidence conclusion, which integrates the different streams of modular information. The assessment method gives higher weight to findings that are mechanistically linked with biological relevance to carcinogenesis. With a focus on EU-REACH, and pending upon successful test method validation and acceptance, this will also enable the MoSt for NGTxC to be applied for regulatory purposes across different regulatory jurisdictions.

Effect of cell treatment procedures on in vitro genotoxicity assessment.

So far, the majority of in vitro toxicological experiments are conducted after an acute 24 h treatment that does not represent a realistic human chemical exposure. Recently, new in vitro approaches have been proposed to study the chemical toxicological effect over several days in order to be more predictive of a representative exposure scenario. In this study, we investigated the genotoxic potential of chemicals (direct or bioactived clastogen, aneugen and apoptotic inducer) with the γH2AX and pH3 biomarkers, in the human liver-derived HepaRP cell line. We used different treatment durations, with or without a three-day recovery stage (release period), before genotoxicity measurement. Data were analysed with the Benchmark Dose approach. We observed that the detection of clastogenic compounds (notably for DNA damaging agents) was more sensitive after three days of repeated treatment compared to one or three treatments over 24 h. In contrast, aneugenic chemicals were detected as genotoxic in a similar manner whether after a 24 h exposure or a three-day repeated treatment. Globally, the release period decreases the genotoxicity measurement substantially. For DNA damaging agents, after high concentration treatments, γH2AX induction was always observed after a three-day release period. In contrast, for DNA topoisomerase inhibitors, no effect could be observed after the release period. In conclusion, in the HepaRP cell line, there are some important differences between a one-day acute and a three-day repeated treatment protocol, indicating that different cell treatment procedures may differentiate chemical genotoxic mechanisms of action more efficiently.

A novel support vector machine-based 1-day, single-dose prediction model of genotoxic hepatocarcinogenicity in rats.

The development of a rapid and accurate model for determining the genotoxicity and carcinogenicity of chemicals is crucial for effective cancer risk assessment. This study aims to develop a 1-day, single-dose model for identifying genotoxic hepatocarcinogens (GHCs) in rats. Microarray gene expression data from the livers of rats administered a single dose of 58 compounds, including 5 GHCs, was obtained from the Open TG-GATEs database and used for the identification of marker genes and the construction of a predictive classifier to identify GHCs in rats. We identified 10 gene markers commonly responsive to all 5 GHCs and used them to construct a support vector machine-based predictive classifier. In the silico validation using the expression data of the Open TG-GATEs database indicates that this classifier distinguishes GHCs from other compounds with high accuracy. To further assess the model's effectiveness and reliability, we conducted multi-institutional 1-day single oral administration studies on rats. These studies examined 64 compounds, including 23 GHCs, with gene expression data of the marker genes obtained via quantitative PCR 24 h after a single oral administration. Our results demonstrate that qPCR analysis is an effective alternative to microarray analysis. The GHC predictive model showed high accuracy and reliability, achieving a sensitivity of 91% (21/23) and a specificity of 93% (38/41) across multiple validation studies in three institutions. In conclusion, the present 1-day single oral administration model proves to be a reliable and highly sensitive tool for identifying GHCs and is anticipated to be a valuable tool in identifying and screening potential GHCs.

Genome-wide surveillance of transcription errors in response to genotoxic stress.

Mutagenic compounds are a potent source of human disease. By inducing genetic instability, they can accelerate the evolution of human cancers or lead to the development of genetically inherited diseases. Here, we show that in addition to genetic mutations, mutagens are also a powerful source of transcription errors. These errors arise in dividing and nondividing cells alike, affect every class of transcripts inside cells, and, in certain cases, greatly exceed the number of mutations that arise in the genome. In addition, we reveal the kinetics of transcription errors in response to mutagen exposure and find that DNA repair is required to mitigate transcriptional mutagenesis after exposure. Together, these observations have far-reaching consequences for our understanding of mutagenesis in human aging and disease, and suggest that the impact of DNA damage on human physiology has been greatly underestimated.

The Srs2 helicase dampens DNA damage checkpoint by recycling RPA from chromatin.

The DNA damage checkpoint induces many cellular changes to cope with genotoxic stress. However, persistent checkpoint signaling can be detrimental to growth partly due to blockage of cell cycle resumption. Checkpoint dampening is essential to counter such harmful effects, but its mechanisms remain to be understood. Here, we show that the DNA helicase Srs2 removes a key checkpoint sensor complex, RPA, from chromatin to down-regulate checkpoint signaling in budding yeast. The Srs2 and RPA antagonism is supported by their numerous suppressive genetic interactions. Importantly, moderate reduction of RPA binding to single-strand DNA (ssDNA) rescues hypercheckpoint signaling caused by the loss of Srs2 or its helicase activity. This rescue correlates with a reduction in the accumulated RPA and the associated checkpoint kinase on chromatin in srs2 mutants. Moreover, our data suggest that Srs2 regulation of RPA is separable from its roles in recombinational repair and critically contributes to genotoxin resistance. We conclude that dampening checkpoint by Srs2-mediated RPA recycling from chromatin aids cellular survival of genotoxic stress and has potential implications in other types of DNA transactions.

Simultaneous and trace-level quantification of four benzene sulfonate potential genotoxic impurities in doxofylline active pharmaceutical ingredients and tablets using high-performance liquid chromatography with ultraviolet detection.

In the production of doxofylline, the common occurrence of toxic p-toluene sulfonate generation prompted the development and validation of a method using HPLC with ultraviolet detection (HPLC-UV). This method is designed for detecting four potential genotoxic impurities (PGIs) present in both doxofylline drug substance and tablets, with a focus on the UV-absorbing group p-toluene sulfonate. The four impurities were methyl 4-methylbenzenesulfonate (PGI-1), ethyl 4-methylbenzenesulfonate (PGI-2), 2-hydroxyethyl 4-methylbenzenesulfonate (PGI-3), and 2-(4-methylphenyl)sulfonyloxyethyl 4-methylbenzenesulfonate (PGI-4). In this method, chromatographic separation was achieved using a Waters Symmetry C18 column (250 mm × 4.6 mm, 5 µm). The mobile phases consisted of 20% acetonitrile as mobile phase A and pure acetonitrile as mobile phase B, operating in gradient elution mode at a flow rate of 1.0 mL/min. According to the guidelines of the International Conference on Harmonization, it was determined that this method could quantify four PGIs at 0.0225 µg/mL in samples containing 60 mg/mL. The validated approach demonstrated excellent linearity (R2 > 0.999) across the concentration range of 30%-200% (relative to 0.075 µg/mL doxofylline) for the four PGIs. The accuracy of this method for the four PGIs ranged from 94.8% to 100.4%. The reverse-phase-HPLC-UV analytical method developed in this study is characterized by its speed and precision, making it suitable for the sensitive analysis of benzene sulfonate PGIs in doxofylline drug substances and tablets.

A sensitive ultra-performance liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of assay and trace-level genotoxic tosylate analogs (methyl and ethyl) in empagliflozin and its tablet dosage forms.

This study performed the simultaneous quantification of assay and two alkyl sulfonate (tosylate) analogs of empagliflozin (EGZ), specifically methyl 4-methyl benzene sulfonate (MMBS) and ethyl 4-methyl benzene sulfonate (EMBS) in EGZ, and its finished dosage form using an accurate and sensitive ultra-performance liquid chromatography-mass spectrometry method. The separation was achieved on a Waters Acquity BEH Shield RP18 (100 × 2.1 mm, 1.7 µm) column in gradient elution mode with 0.1% formic acid and acetonitrile as the mobile phases and a flow rate of 0.5 mL/min. For simultaneous quantification, the multiple reaction monitoring technique was utilized. The procedure was successfully validated in accordance with the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines. The peak areas of both impurities, along with their concentrations, exhibited a good relationship with Pearson's correlation coefficient (R), which was >0.999 in the range of 0.3-6 ppm with an EGZ concentration of 2 mg/mL. The percentage recoveries from the limit of quantitation (LOQ) to 200% to the specification level were in the range of 94.82%-102.92%, whereas the percentage relative standard deviation (%RSD) was <2. Therefore, this method is rapid and accurate to quantify MMBS, EMBS, and EGZ assay simultaneously from the marketed tablet dosage forms of EGZ for commercial release and stability sample testing.

Establishing a multi-method approach for unprecedented detection and quantification of 13 genotoxic impurities (GTIs) in Apixaban drug substance through ultra-performance liquid chromatography (UPLC).

This groundbreaking study introduces a pioneering development of multi-method approach for the first-ever detection and quantification of 13 genotoxic impurities (GTIs) in Apixaban (Apx) drug substance using ultra-performance liquid chromatography (UPLC) with ultraviolet (UV) detector. In this novel endeavor, two distinct UPLC-UV methods, Method A (for impurities A to G) and Method B (for impurities H to M), were meticulously developed and validated as per International Council for Harmonization (ICH) guidelines to address the challenge of identification and control of 13 GTIs in Apx drug substance. The validation process included rigorous assessment of linearity, accuracy, specificity, precision, limit of quantification (LOQ), and limit of detection (LOD) for each impurity in each method which marks a significant advancement in pharmaceutical analysis. The developed methods address the regulatory requirements set forth by ICH M7(R2) guidelines by providing a reliable approach for quantifying GTIs in Apx drug substance at trace levels to minimize the potential carcinogenic risk to the patients.