Quantum Mechanical Assessment of Nitrosamine Potency.

Nitrosamines are in the cohort of concern (CoC) as determined by regulatory guidance. CoC compounds are considered highly potent carcinogens that need to be limited below the threshold of toxicological concern, 1.5 µg/day. Nitrosamines like NDMA and NDEA require strict control, while novel nitrosamine drug substance-related impurities (NDSRIs) may or may not be characterized as potent carcinogens. A risk assessment based on the structural features of NDSRIs is important in order to predict potency because they lack substance-specific carcinogenicity. Herein, we present a quantum mechanical (QM)-based analysis on structurally diverse sets of nitrosamines to better understand how structure influences the reactivity that could result in carcinogenicity. We describe the potency trend through activation energies corresponding to α-hydroxylation, aldehyde formation, diazonium intermediate formation, reaction with DNA base, and hydrolysis reactions, and other probable metabolic pathways associated with the carcinogenicity of nitrosamines. We evaluated activation energies for selected cases such as N-nitroso pyrrolidines, N-nitroso piperidines, N-nitroso piperazines, N-nitroso morpholines, N-nitroso thiomorpholine, N-methyl nitroso aromatic, fluorine-substituted nitrosamines, and substituted aliphatic nitrosamines. We compare these results to the recent framework of the carcinogenic potency characterization approach (CPCA) proposed by health authorities which is meant to give guidance on acceptable intakes (AI) for NDSRIs lacking substance-specific carcinogenicity data. We show examples where QM modeling and CPCA are aligned and examples where CPCA both underestimates and overestimates the AI. In cases where CPCA predicts high potency for NDSRIs, QM modeling can help better estimate an AI. Our results suggest that a combined mechanistic understanding of α-hydroxylation, aldehyde formation, hydrolysis, and reaction with DNA bases could help identify the structural features that underpin the potency of nitrosamines. We anticipate this work will be a valuable addition to the CPCA and provide a more analytical way to estimate AI for novel NDSRIs.

A Kinetic Model for Assessing Potential Nitrosamine Carcinogenicity.

Understanding the potential carcinogenic potency of nitrosamines is necessary to setting acceptable intake limits. Nitrosamines and the components that can form them are commonly present in food, water, cosmetics, and tobacco. The recent observation of nitrosamines in pharmaceuticals highlighted the need for effective methods to determine acceptable intake limits. Herein, we describe two computational models that utilize properties based upon quantum mechanical calculations in conjunction with mechanistic insights and established data to determine the carcinogenic potency of a variety of common nitrosamines. These models can be applied to experimentally untested nitrosamines to aid in the establishment of acceptable intake limits.

Ames test study designs for nitrosamine mutagenicity testing: qualitative and quantitative analysis of key assay parameters.

The robust control of genotoxic N-nitrosamine (NA) impurities is an important safety consideration for the pharmaceutical industry, especially considering recent drug product withdrawals. NAs belong to the 'cohort of concern' list of genotoxic impurities (ICH M7) because of the mutagenic and carcinogenic potency of this chemical class. In addition, regulatory concerns exist regarding the capacity of the Ames test to predict the carcinogenic potential of NAs because of historically discordant results. The reasons postulated to explain these discordant data generally point to aspects of Ames test study design. These include vehicle solvent choice, liver S9 species, bacterial strain, compound concentration, and use of pre-incubation versus plate incorporation methods. Many of these concerns have their roots in historical data generated prior to the harmonization of Ames test guidelines. Therefore, we investigated various Ames test assay parameters and used qualitative analysis and quantitative benchmark dose modelling to identify which combinations provided the most sensitive conditions in terms of mutagenic potency. Two alkyl-nitrosamines, N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) were studied. NDMA and NDEA mutagenicity was readily detected in the Ames test and key assay parameters were identified that contributed to assay sensitivity rankings. The pre-incubation method (30-min incubation), appropriate vehicle (water or methanol), and hamster-induced liver S9, alongside Salmonella typhimurium strains TA100 and TA1535 and Escherichia coli strain WP2uvrA(pKM101) provide the most sensitive combination of assay parameters in terms of NDMA and NDEA mutagenic potency in the Ames test. Using these parameters and further quantitative benchmark dose modelling, we show that N-nitrosomethylethylamine (NMEA) is positive in Ames test and therefore should no longer be considered a historically discordant NA. The results presented herein define a sensitive Ames test design that can be deployed for the assessment of NAs to support robust impurity qualifications.

Detection with NO Modification: (N═O)-Au Interactions for Instantaneous Label-Free Detection of N-Nitrosodiphenylamine.

Increasing concerns have been raised about dangerous, yet nearly undetectable levels of nitrosamines in foods, medications, and drinking water. Their ubiquitous presence and carcinogenicity necessitates a method of sensitive and selective detection of these potent toxins. While the detection of two major nitrosamines─N-nitrosodimethylamine and N-nitrosodiethylamine─has seen success, low detection limits are scarcer for the other members of this class. One member, N-nitrosodiphenylamine (NDPhA), has had little progress not only in its detection in low quantities but also in its detection at all. NDPhA has unique difficulty in its identification due to its aromaticity, making it far more problematic to distinguish in the common GC-MS or LC-MS/MS methods used for nitrosamine sensing. Despite this detection barrier, it has been listed among the top 6 carcinogenic nitrosamines by the Food and Drug Administration as of 2023. Due to its evasive nature, a unique methodology must be applied to facilitate its sensitive identification. Herein, we describe the use of surface-enhanced Raman spectroscopy for the first account of liquid-phase detection of NDPhA using cysteamine-functionalized gold nanostars and a portable Raman spectrometer. Our methodology requires no chemical modification to the nitrosated structure as well as the usage of two well-understood biocompatible materials: cysteamine and gold nanoparticles.

Novel method for monitoring of carcinogenic impurity of N-nitrosamine in nizatidine pharmaceutical products using ultra high-pressure liquid chromatography triple quadrupole mass spectrometry.

Nitrosamine compounds pose a significant concern as potential carcinogens, prompting heightened scrutiny from regulatory bodies, particularly regarding their presence in pharmaceuticals. The detection of unacceptable levels of N-nitrosodiethylamine (NDMA) in ranitidine has led to widespread recalls, driving interest in alternative medications such as nizatidine, which shares a similar pharmacological class and is used to treat various gastrointestinal conditions. Despite fewer reports on NDMA levels in nizatidine, its structural similarity to ranitidine, characterized by a tertiary amine, underscores the potential for NDMA formation. Addressing the analytical challenges associated with nitrosamine detection, this study focuses on developing and validating an ultra-high pressure liquid chromatography triple quadrupole mass spectrometry (UHPLC-MS/MS) method for quantifying NDMA in both nizatidine active pharmaceutical ingredients and tablet formulations. Method validation adheres to International Council for Harmonisation recommendations, with a demonstrated linear range of 0.25-100 ng/mL for NDMA, exhibiting excellent linearity (regression coefficient >0.999) and efficient recovery rates ranging from 95.98% to 109.57%. The method shows high sensitivity, with limits of detection and quantification of 0.25 and 0.5 ng/mL, respectively. The developed UHPLC-MS/MS method offers a simple, precise, accurate, and selective approach for monitoring NDMA levels in nizatidine formulations available in Australia, promising enhanced sensitivity and specificity with limits of quantification in the ppb and sub-ppb ranges.

Role of Carbonyl Compounds for N-Nitrosamine Formation during Nitrosation: Kinetics and Mechanisms.

N-Nitrosamines are potential human carcinogens frequently detected in natural and engineered aquatic systems. This study sheds light on the role of carbonyl compounds in the formation of N-nitrosamines by nitrosation of five secondary amines via different pathways. The results showed that compared to a control system, the presence of formaldehyde enhances the formation of N-nitrosamines by a factor of 5-152 at pH 7, depending on the structure of the secondary amines. Acetaldehyde showed a slight enhancement effect on N-nitrosamine formation, while acetone and benzaldehyde did not promote nitrosation reactions. For neutral and basic conditions, the iminium ion was the dominant intermediate for N-nitrosamine formation, while carbinolamine became the major contributor under acidic conditions. Negative free energy changes (<-19 kcal mol-1) and relatively low activation energies (<18 kcal mol-1) of the reactions of secondary amines with N2O3, iminium ions with nitrite and carbinolamines with N2O3 from quantum chemical computations further support the proposed reaction pathways. This highlights the roles of the iminium ion and carbinolamine in the formation of N-nitrosamines during nitrosation in the presence of carbonyl compounds, especially in the context of industrial wastewater.

Mass Spectrometry-Based Metabolic Profiling of Urinary Metabolites of N'-Nitrosonornicotine (NNN) in the Rat.

The tobacco-specific nitrosamine N'-nitrosonornicotine (NNN) and its close analogue 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) are classified as "carcinogenic to humans" (Group 1) by the International Agency for Research on Cancer. The currently used biomarker to monitor NNN exposure is urinary total NNN (free NNN plus its N-glucuronide). However, total NNN does not provide information about the extent of metabolic activation of NNN as related to its carcinogenicity. Targeted analysis of the major metabolites of NNN in laboratory animals recently led to the identification of N'-nitrosonornicotine-1N-oxide (NNN-N-oxide), a unique metabolite detected in human urine that is specifically formed from NNN. To further investigate NNN urinary metabolites that hold promise as new biomarkers for monitoring NNN exposure, uptake, and/or metabolic activation, we conducted a comprehensive profiling of NNN metabolites in the urine of F344 rats treated with NNN or [pyridine-d4]NNN. Using our optimized high-resolution mass spectrometry (HRMS)-based isotope-labeling method, 46 putative metabolites were identified with robust MS evidence. Out of the 46 candidates, all known major NNN metabolites were identified and structurally confirmed by comparing them to their isotopically labeled standards. More importantly, putative metabolites considered to be exclusively formed from NNN were also identified. The two new representative metabolites─4-(methylthio)-4-(pyridin-3-yl)butanoic acid (23, MPBA) and N-acetyl-S-(5-(pyridin-3-yl)-1H-pyrrol-2-yl)-l-cysteine (24, Py-Pyrrole-Cys-NHAc) ─were identified by comparing them to synthetic standards that were fully characterized by nuclear magnetic resonance and HRMS. They are hypothesized to be formed by NNN α-hydroxylation pathways and thus represent the first potential biomarkers to specifically monitor the uptake plus metabolic activation of NNN in tobacco users.

Computational Prediction of Metabolic α-Carbon Hydroxylation Potential of N-Nitrosamines: Overcoming Data Limitations for Carcinogenicity Assessment.

Recent withdrawal of several drugs from the market due to elevated levels of N-nitrosamine impurities underscores the need for computational approaches to assess the carcinogenicity risk of nitrosamines. However, current approaches are limited because robust animal carcinogenicity data are only available for a few simple nitrosamines, which do not represent the structural diversity of the many possible nitrosamine drug substance related impurities (NDSRIs). In this paper, we present a novel method that uses data on CYP-mediated metabolic hydroxylation of CH2 groups in non-nitrosamine xenobiotics to identify structural features that may also help in predicting the likelihood of metabolic α-carbon hydroxylation in N-nitrosamines. Our approach offers a new avenue for tapping into potentially large experimental data sets on xenobiotic metabolism to improve risk assessment of nitrosamines. As α-carbon hydroxylation is the crucial rate-limiting step in nitrosamine metabolic activation, identifying and quantifying the influence of various structural features on this step can provide valuable insights into their carcinogenic potential. This is especially important considering the scarce information available on factors that affect NDSRI metabolic activation. We have identified hundreds of structural features and calculated their impact on hydroxylation, a significant advancement compared to the limited findings from the small nitrosamine carcinogenicity data set. While relying solely on α-carbon hydroxylation prediction is insufficient for forecasting carcinogenic potency, the identified features can help in the selection of relevant structural analogues in read across studies and assist experts who, after considering other factors such as the reactivity of the resulting electrophilic diazonium species, can establish the acceptable intake (AI) limits for nitrosamine impurities.

From cultivation to cancer: formation of N-nitrosamines and other carcinogens in smokeless tobacco and their mutagenic implications.

Tobacco use is a major cause of preventable morbidity and mortality globally. Tobacco products, including smokeless tobacco (ST), generally contain tobacco-specific N-nitrosamines (TSNAs), such as N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-butanone (NNK), which are potent carcinogens that cause mutations in critical genes in human DNA. This review covers the series of biochemical and chemical transformations, related to TSNAs, leading from tobacco cultivation to cancer initiation. A key aim of this review is to provide a greater understanding of TSNAs: their precursors, the microbial and chemical mechanisms that contribute to their formation in ST, their mutagenicity leading to cancer due to ST use, and potential means of lowering TSNA levels in tobacco products. TSNAs are not present in harvested tobacco but can form due to nitrosating agents reacting with tobacco alkaloids present in tobacco during certain types of curing. TSNAs can also form during or following ST production when certain microorganisms perform nitrate metabolism, with dissimilatory nitrate reductases converting nitrate to nitrite that is then released into tobacco and reacts chemically with tobacco alkaloids. When ST usage occurs, TSNAs are absorbed and metabolized to reactive compounds that form DNA adducts leading to mutations in critical target genes, including the RAS oncogenes and the p53 tumor suppressor gene. DNA repair mechanisms remove most adducts induced by carcinogens, thus preventing many but not all mutations. Lastly, because TSNAs and other agents cause cancer, previously documented strategies for lowering their levels in ST products are discussed, including using tobacco with lower nornicotine levels, pasteurization and other means of eliminating microorganisms, omitting fermentation and fire-curing, refrigerating ST products, and including nitrite scavenging chemicals as ST ingredients.

Comparative Evaluation of Chemical and Photolytic Denitrosation Methods for Chemiluminescence Detection of Total N-Nitrosamines in Wastewater Samples.

N-Nitrosamines form as byproducts during oxidative water treatment and occur as impurities in consumer and industrial products. To date, two methods based on chemiluminescence (CL) detection of nitric oxide liberated from N-nitrosamines via denitrosation with acidic triiodide (HI3) treatment or ultraviolet (UV) photolysis have been developed to enable the quantification of total N-nitrosamines (TONO) in environmental water samples. In this work, we configured an integrated experimental setup to compare the performance of HI3-CL and UV-CL methods with a focus on their applicability for TONO measurements in wastewater samples. With the use of a large-volume purge vessel for chemical denitrosation, the HI3-CL method achieved signal stability and detection limits comparable to those achieved by the UV-CL method which utilized a microphotochemical reactor for photolytic denitrosation. Sixty-six structurally diverse N-nitroso compounds (NOCs) yielded a range of conversion efficiencies relative to N-nitrosodimethylamine (NDMA) regardless of the conditions applied for denitrosation. On average, TONO measured in preconcentrated raw and chloraminated wastewater samples by the HI3-CL method were 2.1 ± 1.1 times those measured by the UV-CL method, pointing to potential matrix interferences as further confirmed by spike recovery tests. Overall, our comparative assessment of the HI3-CL and UV-CL methods serves as a basis for addressing methodological gaps in TONO analysis.

Synthesis and analysis of isotopically stable labeled nitrosamines as mass spectrometry standards for drug impurity quality control.

We describe a simple and easy pathway to synthesize nitrosamine mass spectrometry standards in good to moderate yields. N-alkylation of Boc-protected primary or secondary amines using stable isotope labeled alkyl halides yielded the key intermediates that were deprotected, and then, the nitrosamine was formed with sodium nitrite and sodium hydrogensulfate. Special attention to safety, disposal of waste, and surface cleaning was carried throughout.

Mass Spectrometric Quantitation of N'-Nitrosonornicotine-1N-oxide in the Urine of Cigarette Smokers and Smokeless Tobacco Users.

N'-Nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), which always occur together and are present exclusively in tobacco products, are classified as "carcinogenic to humans" (Group 1) by the International Agency for Research on Cancer. While 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) serves as an excellent biomarker for NNK exposure, the currently available biomarker for NNN exposure is urinary "total NNN" (free NNN plus its N-glucuronide). Quantitation of urinary NNN requires extensive precautions to prevent artifactual formation of NNN resulting from nitrosation of nornicotine during analysis. NNN itself can also be formed endogenously by the same nitrosation reaction, which may sometimes cause an overestimation of exposure to preformed NNN. It is thus important to develop an alternative biomarker to specifically reflect NNN metabolic fate and facilitate relevant cancer etiology studies. In this study, we report the first detection of N'-nitrosonornicotine-1N-oxide (NNN-N-oxide) in human urine. Using a highly specific and sensitive MS3 transition-based method, NNN-N-oxide was quantified with a mean level of 8.40 ± 6.04 fmol/mL in the urine of 10 out of 32 cigarette smokers. It occurred in a substantially higher level in the urine of 13 out of 14 smokeless tobacco users, amounting to a mean concentration of 85.2 ± 96.3 fmol/mL urine. No NNN-N-oxide was detected in any of the nonsmoker urine samples analyzed (n = 20). The possible artifactual formation of NNN-N-oxide during sample preparation steps was excluded by experiments using added ammonium sulfamate. The low levels of NNN-N-oxide in the urine of tobacco users indicate that the pyridine N-oxidation pathway represents a minor detoxification pathway of NNN, which further supports the importance of the α-hydroxylation pathway of NNN metabolic activation in humans.

Thirdhand Exposures to Tobacco-Specific Nitrosamines through Inhalation, Dust Ingestion, Dermal Uptake, and Epidermal Chemistry.

Tobacco-specific nitrosamines (TSNAs) are emitted during smoking and form indoors by nitrosation of nicotine. Two of them, N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), are human carcinogens with No Significant Risk Levels (NSRLs) of 500 and 14 ng day-1, respectively. Another TSNA, 4-(methylnitrosamino)-4-(3-pyridyl) butanal (NNA), shows genotoxic and mutagenic activity in vitro. Here, we present additional evidence of genotoxicity of NNA, an assessment of TSNA dermal uptake, and predicted exposure risks through different pathways. Dermal uptake was investigated by evaluating the penetration of NNK and nicotine through mice skin. Comparable mouse urine metabolite profiles suggested that both compounds were absorbed and metabolized via similar mechanisms. We then investigated the effects of skin constituents on the reaction of adsorbed nicotine with nitrous acid (epidermal chemistry). Higher TSNA concentrations were formed on cellulose and cotton substrates that were precoated with human skin oils and sweat compared to clean substrates. These results were combined with reported air, dust, and surface concentrations to assess NNK intake. Five different exposure pathways exceeded the NSRL under realistic scenarios, including inhalation, dust ingestion, direct dermal contact, gas-to-skin deposition, and epidermal nitrosation of nicotine. These results illustrate potential long-term health risks for nonsmokers in homes contaminated with thirdhand tobacco smoke.

Multiscale computational investigations of the translesion synthesis bypass of tobacco-derived DNA adducts: critical insights that complement experimental biochemical studies.

Among the numerous agents that damage DNA, tobacco products remain one of the most lethal and result in the most diverse set of DNA lesions. This perspective aims to provide an overview of computational work conducted to complement experimental biochemical studies on the mutagenicity of adducts derived from the most potent tobacco carcinogen, namely 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (nicotine-derived nitrosaminoketone or NNK). Lesions ranging from the smallest methylated thymine derivatives to the larger, flexible pyridyloxobutyl (POB) guanine adducts are considered. Insights are obtained from density functional theory (DFT) calculations and molecular dynamics (MD) simulations into the damaged nucleobase and nucleoside structures, the accommodation of the lesions in the active site of key human polymerases, the intrinsic base pairing potentials of the adducts, and dNTP incorporation opposite the lesions. Overall, the computational data provide atomic level information that can rationalize the differential mutagenic properties of tobacco-derived lesions and uncover important insights into the impact of adduct size, nucleobase, position, and chemical composition of the bulky moiety.

Development of a fully automated analytical platform based on static headspace-gas chromatography-tandem mass spectrometry for the analysis of five N-nitrosamines in dried aquatic products of animal origin.

BACKGROUND: The development of rapid and sensitive monitoring methods for trace N-nitrosamines (NAs) in foodstuffs is essential for mitigating the potential health risks to consumers. In the present study, an analytical platform based on one step fully automated static headspace sampling and gas chromatography-tandem mass spectrometry (GC-MS/MS) was developed and validated for the analysis of N-nitrosamines in dried aquatic products of animal origin. The samples and sodium chloride solution mixture were incubated in a heated headspace vial for analyte evaporation, coupled to automatic sampling and online GC-MS/MS analysis. The proposed method requires minimal sample preparation and organic solvent consumption. Five N-nitrosamines including N-nitroso dimethylamine, N-nitroso methyl ethylamine, N-nitroso pyrolidine, N-nitroso piperidine and N-nitroso diphenylamine were selected as model compounds to optimize the significant factors by a using Box-Behnken design. RESULTS: The optimum conditions achieved limits of detections in the range 0.08-0.29 µg kg-1 , with correlation coefficient over 0.998. Relative recoveries in dried aquatic product sample were in the range 76.9-92.4%, with relative SDs of 1.9-7.2%. CONCLUSION: These results confirm the reliability of the developed method for further application in trace level monitoring of the target analytes in foodstuffs. © 2022 Society of Chemical Industry.

Orchestrating Nitric Oxide and Carbon Monoxide Signaling Molecules for Synergistic Treatment of MRSA Infections.

The local delivery of gaseous signaling molecules (GSMs) has shown promising therapeutic potential. However, although GSMs have a subtle interplay in physiological and pathological conditions, the co-delivery of different GSMs for therapeutic purposes remains unexplored. Herein, we covalently graft a nitric oxide (NO)-releasing N-nitrosamine moiety onto the carbon monoxide (CO)-releasing 3-hydroxyflavone (3-HF) antenna, resulting in the first NO/CO-releasing donor. Under visible light irradiation, photo-mediated co-release of NO and CO reveals a superior antimicrobial effect toward Gram-positive bacteria with a combination index of 0.053. The synergy of NO and CO hyperpolarizes and permeabilizes bacterial membranes, which, however, shows negligible hemolysis and no evident toxicity toward normal mammalian cells. Moreover, the co-release of NO and CO can efficiently treat MRSA infection in a murine skin wound model, showing a better therapeutic capacity than vancomycin.

Investigation of 2'-Deoxyadenosine-Derived Adducts Specifically Formed in Rat Liver and Lung DNA by N'-Nitrosonornicotine Metabolism.

The International Agency for Research on Cancer has classified the tobacco-specific nitrosamines N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) as "carcinogenic to humans" (Group 1). To exert its carcinogenicity, NNN requires metabolic activation to form reactive intermediates which alkylate DNA. Previous studies have identified cytochrome P450-catalyzed 2'-hydroxylation and 5'-hydroxylation of NNN as major metabolic pathways, with preferential activation through the 5'-hydroxylation pathway in some cultured human tissues and patas monkeys. So far, the only DNA adducts identified from NNN 5'-hydroxylation in rat tissues are 2-[2-(3-pyridyl)-N-pyrrolidinyl]-2'-deoxyinosine (Py-Py-dI), 6-[2-(3-pyridyl)-N-pyrrolidinyl]-2'-deoxynebularine (Py-Py-dN), and N6-[4-hydroxy-1-(pyridine-3-yl)butyl]-2'-deoxyadenosine (N6-HPB-dAdo) after reduction. To expand the DNA adduct panel formed by NNN 5'-hydroxylation and identify possible activation biomarkers of NNN metabolism, we investigated the formation of dAdo-derived adducts using a new highly sensitive and specific liquid chromatography-nanoelectrospray ionization-high-resolution tandem mass spectrometry method. Two types of NNN-specific dAdo-derived adducts, N6-[5-(3-pyridyl)tetrahydrofuran-2-yl]-2'-deoxyadenosine (N6-Py-THF-dAdo) and 6-[2-(3-pyridyl)-N-pyrrolidinyl-5-hydroxy]-2'-deoxynebularine (Py-Py(OH)-dN), were observed for the first time in calf thymus DNA incubated with 5'-acetoxyNNN. More importantly, Py-Py(OH)-dN was also observed in relatively high abundance in the liver and lung DNA of rats treated with racemic NNN in the drinking water for 3 weeks. These new adducts were characterized using authentic synthesized standards. Both NMR and MS data agreed well with the proposed structures of N6-Py-THF-dAdo and Py-Py(OH)-dN. Reduction of Py-Py(OH)-dN by NaBH3CN led to the formation of Py-Py-dN both in vitro and in vivo, which was confirmed by its isotopically labeled internal standard [pyridine-d4]Py-Py-dN. The NNN-specific dAdo adducts Py-THF-dAdo and Py-Py(OH)-dN formed by NNN 5'-hydroxylation provide a more comprehensive understanding of the mechanism of DNA adduct formation by NNN.

Identification of an N'-Nitrosonornicotine-Specific Deoxyadenosine Adduct in Rat Liver and Lung DNA.

The tobacco-specific nitrosamines N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) are considered to be two of the most important carcinogens in unburned tobacco and its smoke. They readily cause tumors in laboratory animals and are classified as "carcinogenic to humans" by the International Agency for Research on Cancer. DNA adduct formation by these two carcinogens is believed to play a critical role in tobacco carcinogenesis. Among all the DNA adducts formed by NNN and NNK, 2'-deoxyadenosine (dAdo)-derived adducts have not been fully characterized. In the study reported here, we characterized the formation of N6-[4-(3-pyridyl)-4-oxo-1-butyl]-2'-deoxyadenosine (N6-POB-dAdo) and its reduced form N6-PHB-dAdo formed by NNN 2'-hydroxylation in rat liver and lung DNA. More importantly, we characterized a new dAdo adduct N6-[4-hydroxy-1-(pyridine-3-yl)butyl]-2'-deoxyadenosine (N6-HPB-dAdo) formed after NaBH3CN or NaBH4 reduction both in vitro in calf thymus DNA reacted with 5'-acetoxy-N'-nitrosonornicotine and in vivo in rat liver and lung upon treatment with NNN. This adduct was specifically formed by NNN 5'-hydroxylation. Chemical standards of N6-HPB-dAdo and the corresponding isotopically labeled internal standard [pyridine-d4]N6-HPB-dAdo were synthesized using a four-step method. Both NMR and high-resolution mass spectrometry data agreed well with the proposed structure of N6-HPB-dAdo. The new adduct coeluted with the synthesized internal standard under various LC conditions. Its product ion patterns of MS2 and MS3 transitions were also consistent with the proposed fragmentation patterns. Chromatographic resolution of the two diastereomers of N6-HPB-dAdo was successfully achieved. Quantitation suggested a dose-dependent response of the levels of this new adduct in the liver and lung of rats treated with NNN. However, its level was lower than that of 2-[2-(3-pyridyl)-N-pyrrolidinyl]-2'-deoxyinosine, a previously reported dGuo adduct that is also formed from NNN 5'-hydroxylation. The identification of N6-HPB-dAdo in this study leads to new insights pertinent to the mechanism of carcinogenesis by NNN and to the development of biomarkers of NNN metabolic activation.

Synthesis, anti-microbial, toxicity and molecular docking studies of N-nitroso-N-phenylhydroxylamine (cupferron) and its derivatives.

Bacterial resistance to antimicrobial agents is increasing at an alarming rate globally and requires new lead compounds for antibiotics. In this study, N-phenyl-N-nitroso hydroxylamine (cupferron) and its derivatives have been synthesised using readily available starting materials. The compounds were obtained in high yield and purity. They show activity towards a range of Gram-positive and Gram-negative pathogenic bacteria, with minimum inhibitory concentration (MIC) values as low as 2 µg.mL-1 against the tested organisms, especially for Gram-positive species. Toxicity studies on the lead compound 3b indicate insignificant effects on healthy cell lines. Molecular docking studies on the lead compound identify possible binding modes of the compound, and the results obtained correlate with those of in vitro and MIC studies. The lead compound shows excellent drug-likeness properties.

E-cigarette smoke damages DNA and reduces repair activity in mouse lung, heart, and bladder as well as in human lung and bladder cells.

E-cigarette smoke delivers stimulant nicotine as aerosol without tobacco or the burning process. It contains neither carcinogenic incomplete combustion byproducts nor tobacco nitrosamines, the nicotine nitrosation products. E-cigarettes are promoted as safe and have gained significant popularity. In this study, instead of detecting nitrosamines, we directly measured DNA damage induced by nitrosamines in different organs of E-cigarette smoke-exposed mice. We found mutagenic O6-methyldeoxyguanosines and γ-hydroxy-1,N2 -propano-deoxyguanosines in the lung, bladder, and heart. DNA-repair activity and repair proteins XPC and OGG1/2 are significantly reduced in the lung. We found that nicotine and its metabolite, nicotine-derived nitrosamine ketone, can induce the same effects and enhance mutational susceptibility and tumorigenic transformation of cultured human bronchial epithelial and urothelial cells. These results indicate that nicotine nitrosation occurs in vivo in mice and that E-cigarette smoke is carcinogenic to the murine lung and bladder and harmful to the murine heart. It is therefore possible that E-cigarette smoke may contribute to lung and bladder cancer, as well as heart disease, in humans.