4-(Methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its major metabolite 4-(methylnitrosamino)-l-(3-pyridine)-l-butanol (NNAL) are tobacco-specific lung carcinogens. Methods have been developed to quantify NNK- and...4-(Methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its major metabolite 4-(methylnitrosamino)-l-(3-pyridine)-l-butanol (NNAL) are tobacco-specific lung carcinogens. Methods have been developed to quantify NNK- and NNAL-specific DNA adducts in preclinical samples but are not feasible to translation due to limited access to target tissues for sufficient DNA. In addition, NNAL-specific DNA or protein adducts have never been detected in clinical samples, which are critical to assess the physiological relevance of NNAL bioactivation and carcinogenesis. We herein reported a highly sensitive and specific LC-MS/MS method to quantify the hydrolyzed product, 1-(3-pyridyl)-1,4-butanediol (PBD), from NNAL-induced protein adduct. This method was applied to a variety of biological samples to assess tobacco exposure and NNAL bioactivation.
Secondary organic aerosol (SOA) accounts for a large fraction of fine particulate matter (PM) in the atmosphere. Epidemiological studies have shown that SOA has adverse effects on human health. However, the current knowl...Secondary organic aerosol (SOA) accounts for a large fraction of fine particulate matter (PM) in the atmosphere. Epidemiological studies have shown that SOA has adverse effects on human health. However, the current knowledge of the SOA's effect on the nervous system remains poorly understood. To address this issue, PC12 cells were incubated in SOA from α-pinene ozonation. The results showed that concentration-dependent increases in reactive oxygen species (ROS) levels lead to a decrease in cell viability, indicating that SOA could induce apoptosis and oxidative stress in cells. The peroxides present in the SOA are identified as major contributors to the apoptotic effect. Furthermore, the apoptosis mechanism was analyzed by Western blotting, revealing activation of the mitochondria-associated Bax/Bcl-2-Caspase-3-PARP signal pathway. In addition, the qPCR result showed that SOA had altered the expression of inflammatory factors, including IL-6, IL-1β, and TNF-α, in PC12 cells. This study investigates the molecular-level evidence of the toxicological impact of SOA on the nervous system, which further evaluates the effects of SOA on health.
Silver nanoparticles (AgNPs), a promising class of metallic nanomaterials with strong antibacterial properties and biomedical potential, are increasingly being used in a variety of consumer products. The widespread appli...Silver nanoparticles (AgNPs), a promising class of metallic nanomaterials with strong antibacterial properties and biomedical potential, are increasingly being used in a variety of consumer products. The widespread application of AgNPs has raised concerns about their toxicological effects, particularly their accumulation in the liver and the associated oxidative stress. However, the precise molecular mechanisms driving these effects remain unclear. In this study, we provide evidence that AgNPs trigger ferroptosis in both mouse hepatocytes and HepG2 cells. Transcriptomic analysis identified ferroptosis is a primary cellular response to AgNP exposure, with Nrf2 serving a protective function. Specifically, AgNPs increased p62 expression, which in turn stabilized Nrf2 by suppressing its interaction with Keap1. Upon activation, Nrf2 enhances the transcription of key antioxidant enzymes, including NQO1 and HO-1, thereby alleviating ferroptosis. Additionally, we discovered that Nrf2 activation regulates iron storage by modulating FTH and FTL expression, thereby mitigating AgNP-induced ferroptosis in hepatocytes. These findings clarify the molecular basis of AgNP-induced ferroptosis in hepatocytes and underscore the crucial role of Nrf2 signaling in counteracting oxidative stress and ferroptosis.
Glattke TJ, Mojica MA, Cottrill KA
… +4 more, Lagon SR, Ruto B, Hill D, Cunningham BR
Chem Res Toxicol
· 2025 Dec · PMID 40833067
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Harmful cyanobacterial blooms (HCBs) are a public health concern and require ongoing surveillance to monitor the negative water quality effects and cyanotoxins associated with these blooms. (+)-Anatoxin-a (ATX) is a pote...Harmful cyanobacterial blooms (HCBs) are a public health concern and require ongoing surveillance to monitor the negative water quality effects and cyanotoxins associated with these blooms. (+)-Anatoxin-a (ATX) is a potent neurotoxin produced by select cyanobacteria during HCB formation. Many HCB toxins are commonly associated with discolored water; however, ATX can be present in clear water, which results in a high risk of exposure by accidental ingestion for humans and animals. In this work, we used a qualitative, semitargeted liquid chromatography high resolution mass spectrometry (LC-HRMS) method and a discovery data analysis workflow to detect and identify ATX and its predicted mammalian metabolites in urine samples from ATX-dosed mice. Potential compounds were evaluated for identification with product-ion spectral matching to a local library, accurate mass list matching, further data processing and interpretation, and comparison to undosed mice urine samples. As a result, ATX and dihydroanatoxin-a (dhATX) were successfully identified in the dosed mice samples through retention time (RT) and product-ion spectral matching to their respective commercial standards. The positive identification of dhATX suggests its formation as an abundant metabolic product of ATX within mammalian systems. Additionally, multiple chromatographic peaks were observed that matched the exact mass of 3-OH ATX and were further identified by the presence of diagnostic product ions and comparison to a standard synthesized in-house. In total, seven potential ATX metabolites, including dhATX and 3-OH ATX, were detected and characterized in the dosed mice samples. All identified metabolites were either oxidized or reduced forms of ATX, which suggests that oxidation and reduction are the main pathways for endogenous ATX metabolism in mice. These results are among the first reports of metabolic products of ATX in biological samples and provide a metabolic profile of ATX for higher confidence screening for ATX after a suspected exposure event.
Modaresi SMS, Becanova J, Vojta S
… +8 more, Ryu S, Kaye EM, Agudelo J, Diolintzi A, Skende O, Storch J, Fischer FC, Slitt A
Chem Res Toxicol
· 2025 Sep · PMID 40828089
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Perfluorooctanesulfonate (PFOS) is a persistent environmental pollutant in the per- and polyfluoroalkyl substances (PFAS) class, known to accumulate in the liver and trigger hepatotoxicity. While studies suggested that...Perfluorooctanesulfonate (PFOS) is a persistent environmental pollutant in the per- and polyfluoroalkyl substances (PFAS) class, known to accumulate in the liver and trigger hepatotoxicity. While studies suggested that fatty acid-binding proteins (FABPs) drive the hepatic accumulation of PFAS, evidence is entirely lacking. Using wild-type and mice with global deletion of liver-type and intestine-type FABP (L-FABP, I-FABP), we measured PFOS toxicokinetics by administering single oral doses (0.1, 0.5, and 5 mg/kg) and tracking blood and excreta levels for 65 days. PFOS levels in various tissues were measured at test end. Additionally, we measured PFAS binding to liver tissues from wild-type and FABP knockout mice. Contrary to previous findings, FABP deletion did not significantly alter PFOS blood concentrations, tissue distribution, or elimination rates. Elimination half-lives, clearances, and volumes of distribution were consistent across genotypes, suggesting that neither L-FABP nor I-FABP are critical drivers for PFOS toxicokinetics. binding assays showed similar liver partition coefficients between wild-type and knockout livers for 15 of 19 PFAS, with small differences for some sulfonamides and fluorotelomer sulfonates. These results challenge the presumed role of L-FABP and/or I-FABP in PFAS toxicokinetics, highlighting the need to explore alternative toxicokinetic mechanisms─such as phospholipid binding and transporter-mediated uptake─driving PFAS distribution and elimination.
Composites are popular materials for, among others, restorative dentistry because of their favorable mechanical and esthetic properties and direct-filling applications. The raw materials for such composites usually consi...Composites are popular materials for, among others, restorative dentistry because of their favorable mechanical and esthetic properties and direct-filling applications. The raw materials for such composites usually consist of filler particles embedded in a matrix of dimethacrylate monomers that are polymerized . Because the raw materials cannot polymerize completely, residual monomers leach out over time. The conjugation of methacrylates with sulfur compounds has been recognized as an important reaction as well as a detoxification pathway; thus, leached monomers are expected to undergo chemical reactions with various biomolecules that contain thiol functionalities. To understand the reaction of dental methacrylate monomers with thiols, we studied the reaction of 2-hydroxyethyl methacrylate (HEMA), triethylene glycol dimethacrylate, urethane dimethacrylate, and bisphenol A diglycidyl methacrylate with the model thiol 2-mercaptoethanol using liquid chromatography coupled to low- and high-resolution mass spectrometry (LC-MS and LC-HRMS). The results indicate that thiols react readily with the conjugated double bond, and with methacrylate half-lives of 7-21 h under pseudo-first-order reaction conditions and at neutral pH. Dimethacrylates first formed a monoaddition product, while thiol addition to the second acrylate moiety was observed on a longer time scale. The reaction of HEMA with l-cysteine and l-glutathione was studied in more detail using HRMS and NMR spectroscopy. The reaction rates were substantially higher than for the reaction with mercaptoethanol, and NMR analysis revealed the presence of two isomeric reaction products. Structural characterization also included the identification and assignment of sulfoxides of HEMA-cysteine and HEMA-glutathione. Using the characterized HEMA-thiols as reference standards for LC-HRMS, we demonstrated the presence of HEMA-glutathione, HEMA-cysteine, their sulfoxides, and a putative HEMA-cysteinylglycine in a human osteoblast-like cell line following exposure to HEMA.
Per- and poly fluoroalkyl substances (PFAS) have become a global concern due to their persistence in the environment, contaminating drinking water, air, and soil. Human exposure to PFAS can potentially cause adverse effe...Per- and poly fluoroalkyl substances (PFAS) have become a global concern due to their persistence in the environment, contaminating drinking water, air, and soil. Human exposure to PFAS can potentially cause adverse effects due to its bioaccumulation and nonbiodegradability. To fully understand the role of PFAS in human health conditions, it is important to elucidate their roles in cellular toxicity and biotransformation pathways. Noncovalent complexation of PFAS to proteins is one potential mode of toxicity that can be investigated by comparing structural differences between native and bound proteins. In this work, we perform collision-induced unfolding (CIU) using a cyclic ion mobility-mass spectrometer (cIM-MS) to measure the effects of PFAS binding on protein structure. CIU characterizes the unfolding pathway of analytes by measuring changes in analyte size and shape as a function of increasing activation energy. The CIU results of different species can then be compared to determine potential structural changes. This method is demonstrated using ubiquitin as a model protein and three related PFAS: perfluorobutanesulfonic acid (PFBS), perfluorohexanesulfonic acid (PFHxS), and perfluorooctanesulfonic acid (PFOS). All three PFAS have the same sulfonate headgroup but different fluorinated chain lengths. We observed both qualitative and quantitative differences in ubiquitin unfolding based on the number of bound PFAS molecules as well as the PFAS chain length, suggesting that these molecules are not necessarily passive when associated with protein. Primarily, our results demonstrate a rapid, targeted analysis that can characterize the noncovalent complexation of toxins to biological molecules.
Chemicals may cause cardiotoxicity by binding to the K channel encoded by the human -related gene (hERG). Given the ever-increasing number of chemicals, developing models to efficiently fill the hERG binding affinity da...Chemicals may cause cardiotoxicity by binding to the K channel encoded by the human -related gene (hERG). Given the ever-increasing number of chemicals, developing models to efficiently fill the hERG binding affinity data gap is more desirable than conducting time-consuming experimental tests. However, previous data sets with limited chemical space hindered the development of models with high prediction accuracy and broad applicability domains (ADs). Herein, an expanded hERG binding affinity data set containing diverse categories of chemicals was constructed and subsequently employed to develop machine learning models. ADs of the constructed models were defined by an innovative structure-activity landscape (SAL)-based AD characterization (AD), which considers activity cliffs within SALs formed by molecules with similar structures but inconsistent bioactivities. The optimal model constrained by the AD achieved a coefficient of determination up to 0.89 on the external-validation set, which significantly outperformed previous models. The model coupled with the AD constraint was applied to predict hERG binding affinities for more than 100,000 chemicals from multiple inventories, identifying over 5,000 potential hERG blockers. The model with AD can serve as an efficient and reliable tool for bridging the hERG-mediated cardiotoxicity data vacancy to support sound chemical management.
Ammonium perfluoro (2-methyl-3-oxahexanoate) (GenX), a substitute for perfluorooctanoic acid, disrupts early-life intestinal homeostasis and impacts neurodevelopment. However, the mechanisms are unclear, and intervention...Ammonium perfluoro (2-methyl-3-oxahexanoate) (GenX), a substitute for perfluorooctanoic acid, disrupts early-life intestinal homeostasis and impacts neurodevelopment. However, the mechanisms are unclear, and interventions are limited. In this study, pregnant mice were exposed to GenX (2 mg/kg/day) and chlorogenic acid (CGA, 30 mg/kg/day) from gestation day 0 to postnatal day 21. GenX exposure resulted in a significant reduction in birth length, body weight, and colon length in the pups as well as an infiltration of inflammatory cells, glandular atrophy, and a decrease in the number of goblet cells within the colon. Moreover, the expression of ZO-1, occludin, and claudin-5 decreased in the colon, indicating that exposure to GenX may have compromised intestinal barrier function. The GenX group exhibited increased levels of lipopolysaccharide (LPS) in both the serum and cortex, along with increased expression of NLRP3, GSDMD, GSDMD-N, IL-1β, IL-18, and Caspase-1 p10 in the colon and cortex, indicating pyroptosis activation. The elevated protein expression levels of inflammatory factors, including TNF-α, IFN-γ, COX-2, iNOS, p-PI3K, p-AKT, and p-NF-κB in the cortex, indicated the activation of the PI3K/AKT/NF-κB signaling pathway, contributing to the developmental neurotoxicity. CGA treatment improved intestinal barrier function and reduced LPS leakage and inflammation in the cortex, possibly by decreasing LPS translocation and pyroptosis. Taken together, CGA treatment effectively alleviated perinatal GenX exposure-induced intestinal homeostasis disruption and developmental neurotoxicity due to the LPS translocation and activation of pyroptosis.
Triclocarban (TCC) is an antiseptic ingredient incorporated into many skin-contact hygiene products, raising public health concerns for its frequent detection in the general population. As the central metabolic organ, th...Triclocarban (TCC) is an antiseptic ingredient incorporated into many skin-contact hygiene products, raising public health concerns for its frequent detection in the general population. As the central metabolic organ, the liver plays a key role in lipid synthesis and metabolism; however, the in vivo effects of TCC on hepatic lipid homeostasis remain largely unclear. Herein, a percutaneous TCC exposure model was established based on human-relevant concentrations. An integrated multiomics approach, including hepatic transcriptomics and lipidomics, was applied to explore TCC effects on the liver. We discovered that continuous dermal absorption of TCC significantly disturbed hepatic lipid profiles, as manifested by the decrease in energy storage lipid triacylglycerol (TG) and its synthetic precursor diacylglycerol (DG). Integrated analysis of transcriptomics and targeted validation revealed that TG reduction could result from the decline in lipogenesis, acceleration of fatty acid β-oxidation, and elevated secretion of very-low-density lipoproteins (VLDLs). Cell membrane homeostasis was also disrupted through altering hepatocellular phosphatidylcholine (PC) and phosphatidylethanolamine (PE) levels, which may be related to the activation of endoplasmic reticulum (ER) stress, resulting in the promotion of hepatocyte apoptosis. Together, this work provides novel insights into the causal relationship between TCC exposure and the hepatic metabolic homeostasis.
Breast cancer resistance protein (BCRP), an important ATP-binding cassette transporter, is mainly responsible for drug efflux from cells, especially in high-expressing tumor cells, and is closely associated with multidru...Breast cancer resistance protein (BCRP), an important ATP-binding cassette transporter, is mainly responsible for drug efflux from cells, especially in high-expressing tumor cells, and is closely associated with multidrug resistance (MDR). Numerous studies have demonstrated that the inhibition of BCRP can reverse MDR, so inhibiting BCRP is considered to be a promising strategy for cancer treatment. Alkaloids are the primary bioactive ingredients in various traditional Chinese medicines (TCMs), some of which have been reported to reverse MDR by inhibiting BCRP. Our objective was to identify potential inhibitors of BCRP from 130 alkaloids, evaluate the reversion of MDR in TMZ-resistant U251T and T98G cells, and clarify the structure-activity relationships of alkaloids in BCRP inhibition. Among them, eight alkaloids, including sempervirine, reserpine, coptisine chloride, geissoschizine methyl ether, vincristine sulfate, tetrahydroberberine, cyclovirobuxine, and berberrubine, exhibited significant inhibition (>50%) of BCRP in BCRP-MDCK cells, with IC ranging from 16.95-94.13 μM. Co-treatment with the inhibitor increased Temozolomide (TMZ) cytotoxicity in TMZ-resistant U251T and T98G cells, with IC values declining by 2.1-97.3%. For sempervirine, coptisine chloride, and reserpine, the inhibition appeared to be even greater than the positive inhibitor KO143. Molecular docking analyses elucidated that the inhibitory effect of alkaloids on BCRP was related to π-π stacked, π-alkyl, and π-Sulfur interactions. The pharmacophore model illustrated that aromatic rings and hydrophobic groups may play a critical role in the potency of alkaloid inhibition on BCRP. Taken together, our findings provide valuable information for optimizing alkaloid structure and developing BCRP inhibitors with improved potency and specificity to reverse clinical MDR.
Cisplatin (DDP) is widely utilized in the clinical treatment of malignant tumors, but its effectiveness is significantly compromised by the adverse effects of acute kidney injury (AKI). Renal tubular cells are primarily...Cisplatin (DDP) is widely utilized in the clinical treatment of malignant tumors, but its effectiveness is significantly compromised by the adverse effects of acute kidney injury (AKI). Renal tubular cells are primarily responsible for DDP-induced AKI (DDP-AKI); however, the responses of heterogeneous renal tubular cells to DDP exposure have not been thoroughly explored. In this study, we employed a targeted metabolomics approach to investigate the metabolic responses of renal tubular cells in DDP-AKI rats. Tubular cells were isolated from the renal cortex and outer medulla, and a chemical derivatization-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) metabolomics method was applied. Our findings revealed distinct metabolic profiles in tubular cells from the renal cortex and outer medulla, with outer medullary cells exhibiting greater sensitivity to DDP exposure. Further analyses identified the tryptophan pathway as a critical factor contributing to these regional differences. Additional functional investigations showed that intermediate metabolites of the tryptophan pathway alleviated DDP cytotoxicity in both cortical and outer medullary tubular cells primarily through modulation of the Bcl2/Bax and Caspase-3 pathway. This study enhances our understanding of the metabolic characteristics of tubular cells across heterogeneous renal regions in DDP-AKI and facilitates further exploration of the underlying mechanisms of DDP-induced nephrotoxicity.
Kapiamba KF, Chuang HY, Hao W
… +4 more, Lin TC, Chen LC, Huang YW, Wang Y
Chem Res Toxicol
· 2025 Aug · PMID 40771151
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The rapid proliferation of electronic cigarettes (ECs) has raised significant concerns about their potential health effects on both users and bystanders. This study systematically investigates the impact of EC aerosol ex...The rapid proliferation of electronic cigarettes (ECs) has raised significant concerns about their potential health effects on both users and bystanders. This study systematically investigates the impact of EC aerosol exposure on human alveolar epithelial cells (A549), considering variations in device parameters, nicotine concentration, and exposure type. Using a gravity-based air-liquid interface exposure system, we assessed cytotoxicity and epithelial barrier integrity by measuring cell viability and transepithelial electrical resistance (TEER). Our results indicate that EC aerosol exposure significantly reduces cell viability and disrupts monolayer integrity in a dose- and device-dependent manner. Notably, VUSE (pod-type) exposure led to a 16% decrease in viability and a 41% reduction in TEER, while VOOPOO (mod-type) exposure caused a 25% viability loss and a 61% reduction in TEER. Power settings played a critical role: at 60 W, cell viability dropped by 48% at 12 mg/mL nicotine concentration compared to 29% at 0 mg/mL. Moreover, under the same number of puffs (30 puffs), firsthand exposure resulted in a 73% viability decrease, whereas secondhand exposure showed a 47% reduction, indicating substantial bystander risks associated with EC usage. These findings underscore the importance of device specifications and exposure conditions in determining EC aerosol toxicity. The observed epithelial barrier disruption suggests increased vulnerability to respiratory diseases. Given the comparable toxicity of firsthand and secondhand aerosols, regulatory measures should extend beyond direct users to include bystander protection. This study highlights the urgent need for comprehensive toxicity assessments to inform public health policies on EC use.
Chem Res Toxicol
· 2025 Aug · PMID 40765284
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The toxicological interpretation of metabolomics data remains challenging, mainly due to the lack of relational knowledge of metabolic pathway perturbations and adverse outcomes. Here we propose an approach focused on th...The toxicological interpretation of metabolomics data remains challenging, mainly due to the lack of relational knowledge of metabolic pathway perturbations and adverse outcomes. Here we propose an approach focused on the associative events defined by the adverse outcome pathway (AOP) concept to derive adverse effect predictions from toxicology metabolomics data sets by combining knowledge-driven hypothesis generation and data-driven hypothesis testing. By assessing the associative key events in an AOP, a list of plausible metabolite perturbations can be created, aiding the interpretation of the list of observed metabolite perturbations or differentially abundant metabolites (DAMs). We describe the critical steps of the interpretation and certainty assessment of the effect prediction using protoporphyrinogen oxidase (PPO) inhibition as an example. The approach could serve as a stepping stone toward creating a database of validated, toxicologically meaningful associative event signatures that can be deployed both in (early stage) research of chemical product development and in regulatory chemical safety assessment for hazard identification.
Feng J, Liu CW, Peng J
… +4 more, Hsiao YC, Chen D, Jin C, Lu K
Chem Res Toxicol
· 2025 Aug · PMID 40742251
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Formaldehyde (FA) is a pervasive environmental organic pollutant and a Group 1 human carcinogen. While FA has been implicated in various cancers, its genotoxic effects, including DNA damage and DNA-protein cross-linking,...Formaldehyde (FA) is a pervasive environmental organic pollutant and a Group 1 human carcinogen. While FA has been implicated in various cancers, its genotoxic effects, including DNA damage and DNA-protein cross-linking, have proven insufficient to fully explain its role in carcinogenesis, suggesting the involvement of epigenetic mechanisms. Histone post-translational modifications (PTMs) on H3 and H4, which are critical for regulating gene expression, may contribute to FA-induced pathogenesis, as lysine and arginine residues serve as targets for FA-protein adduct formation. This study aimed to elucidate the epigenetic consequences of FA on histone methylation and acetylation patterns through a comprehensive peptide analysis. Human bronchial epithelial cells (BEAS-2B) were exposed to low-dose (0.1 mM) and high-dose (0.5 mM) FA for 1 h, and their histone extracts were analyzed using high-resolution liquid chromatography-tandem mass spectrometry-based proteomics followed by PTM-combined peptide analysis and single PTM site/type comparisons. We identified 40 peptides on histone H3 and 16 on histone H4 bearing epigenetic marks. Our findings revealed that FA exposure induced systemic alterations in H3 and H4 methylation and acetylation, including hypomethylation of H3K4 and H3K79; changes in H3K9, H3K14, H3K18, H3K23, H3K27, H3K36, H3K37, and H3R40; as well as modifications in H4K5, H4K8, H4K12, and H4K16. These FA-induced histone modifications exhibited strong parallels with epigenetic alterations observed in cancers, leukemia, and Alzheimer's disease. This study provides novel evidence of FA-induced epigenetic toxicity, offering new insights into the potential mechanisms underlying FA-driven pathogenesis.
Chem Res Toxicol
· 2025 Oct · PMID 40719316
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DNA-peptide cross-links (DpCs) are generated via the proteolytic cleavage of DNA-protein cross-links (DPCs), ubiquitous DNA lesions that block DNA replication and transcription. Translesion synthesis (TLS) DNA polymerase...DNA-peptide cross-links (DpCs) are generated via the proteolytic cleavage of DNA-protein cross-links (DPCs), ubiquitous DNA lesions that block DNA replication and transcription. Translesion synthesis (TLS) DNA polymerases can facilitate replication bypass of DpC adducts in either an error-free or error-prone manner. We have previously demonstrated that local DNA sequence context significantly influences hPol -mediated replication bypass of 5-formylcytosine (5fC)-mediated DpC lesions. However, the effects of peptide sequence on the efficiency and fidelity of the TLS bypass of 5fC-mediated DpC lesions remained unknown. In the present study, model DpCs containing three different peptides (NH-GGGKGLGGGA-COOH, NH-RPPQQFFGLM-COOH, and NH-RPKPQQFGLM-COOH, = oxy-lysine) were subjected to primer extension experiments in the presence of TLS polymerases. We found that replication of DpC-containing templates by hPol was more efficient than that catalyzed by hPol or hPol κ. HPLC-ESI-MS and HPLC-ESI-MS/MS analyses of hPol primer extension products indicated that the replication bypass of DpC containing NH-RP*PQQFFGLM-COOH was more error-prone than replication of the other two DpCs, leading to targeted C → T transitions, small deletions, and untargeted mutations downstream from the lesion. Steady-state kinetics investigation of hPol -catalyzed nucleotide incorporation opposite the DpC lesions containing three different peptides revealed that, in all cases, error-free replication was far more efficient than incorporation of incorrect nucleotides. For mutagenic bypass, the catalytic efficiency of hPol -mediated dAMP misincorporation opposite DpC with peptide NH-RPPQQFFGLM-COOH was higher than adenine misincorporation across from the other two DpCs and unmodified dC. These steady-state kinetic findings were further explained by molecular modeling and molecular dynamics simulations, revealing that the three different DpC lesions impose varying perturbations to the geometry of the C-G and C-A pairs at the hPol active site. Collectively, our results reveal that the peptide sequence and conjugation chemistry of DpC lesions can influence the fidelity of lesion bypass by TLS polymerases.