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.

Exposome Profiling of Environmental Pollutants in Seminal Plasma and Novel Associations with Semen Parameters.

Indicators of male fertility are in decline globally, but the underlying causes, including the role of environmental exposures, are unclear. This study aimed to examine organic chemical pollutants in seminal plasma, including both known priority environmental chemicals and less studied chemicals, to identify uncharacterized male reproductive environmental toxicants. Semen samples were collected from 100 individuals and assessed for sperm concentration, percent motility, and total motile sperm. Targeted and nontargeted organic pollutant exposures were measured from seminal plasma using gas chromatography, which showed widespread detection of organic pollutants in seminal plasma across all exposure classes. We used principal component pursuit (PCP) on our targeted panel and derived one component (driven by etriadizole) associated with total motile sperm (p < 0.001) and concentration (p = 0.03). This was confirmed by the exposome-wide association models using individual chemicals, where etriadizole was negatively associated with total motile sperm (FDR q = 0.01) and concentration (q = 0.07). Using PCP on 814 nontargeted spectral peaks identified a component that was associated with total motile sperm (p = 0.001). Bayesian kernel machine regression identified one principal driver of this association, which was analytically confirmed to be N-nitrosodiethylamine. These findings are promising and consistent with experimental evidence showing that etridiazole and N-nitrosodiethylamine may be reproductive toxicants.

Role of TLR4 signaling pathway in the mitigation of damaged lung by low-dose gamma irradiation.

Organisms frequently suffer negative effects from large doses of ionizing radiation. However, radiation is not as hazardous at lower doses as was once believed. The current study aims to evaluate the possible radio-adaptive effect induced by low-dose radiation (LDR) in modulating high-dose radiation (HDR) and N-nitrosodiethylamine (NDEA)-induced lung injury in male albino rats. Sixty-four male rats were randomly divided into four groups: Group 1 (control): normal rats; Group 2 (D): rats given NDEA in drinking water; Group 3 (DR): rats administered with NDEA then exposed to fractionated HDR; and Group 4 (DRL): rats administered with NDEA then exposed to LDR + HDR. In the next stage, malondialdehyde (MDA), glutathione reduced (GSH), catalase (CAT), and superoxide dismutase (SOD) levels in the lung tissues were measured. Furthermore, the enzyme-linked immunoassay analysis technique was performed to assess the Toll-like receptor 4 (TLR4), interleukin-1 receptor-associated kinase 4 (IRAK4), and mitogen-activated protein kinases (MAPK) expression levels. Histopathological and DNA fragmentation analyses in lung tissue, in addition to hematological and apoptosis analyses of the blood samples, were also conducted. Results demonstrated a significant increase in antioxidant defense and a reduction in MDA levels were observed in LDR-treated animals compared to the D and DR groups. Additionally, exposure to LDR decreased TLR4, IRAK4, and MAPK levels, decreased apoptosis, and restored all the alterations in the histopathological, hematological parameters, and DNA fragmentation, indicating its protective effects on the lung when compared with untreated rats. Taken together, LDR shows protective action against the negative effects of subsequent HDR and NDEA. This impact may be attributable to the adaptive response induced by LDR, which decreases DNA damage in lung tissue and activates the antioxidative, antiapoptotic, and anti-inflammatory systems in the affected animals, enabling them to withstand the following HDR exposure.

An update on the current status and prospects of nitrosation pathways and possible root causes of nitrosamine formation in various pharmaceuticals.

Over the last two years, global regulatory authorities have raised safety concerns on nitrosamine contamination in several drug classes, including angiotensin II receptor antagonists, histamine-2 receptor antagonists, antimicrobial agents, and antidiabetic drugs. To avoid carcinogenic and mutagenic effects in patients relying on these medications, authorities have established specific guidelines in risk assessment scenarios and proposed control limits for nitrosamine impurities in pharmaceuticals. In this review, nitrosation pathways and possible root causes of nitrosamine formation in pharmaceuticals are discussed. The control limits of nitrosamine impurities in pharmaceuticals proposed by national regulatory authorities are presented. Additionally, a practical and science-based strategy for implementing the well-established control limits is notably reviewed in terms of an alternative approach for drug product N-nitrosamines without published AI information from animal carcinogenicity testing. Finally, a novel risk evaluation strategy for predicting and investigating the possible nitrosation of amine precursors and amine pharmaceuticals as powerful prevention of nitrosamine contamination is addressed.

Anticancer therapeutic potential of phosphorylated galactosylated chitosan against N-nitrosodiethyl amine-induced hepatocarcinogenesis.

Chitosan is a natural polyfunctional polymer that can be modified to achieve compounds with tailored properties for targeting and treating different cancers. In this study, we report the development and anticancer potential of phosphorylated galactosylated chitosan (PGC). The synthesized compound was characterized by FT-IR, NMR, and mass spectroscopy. The interaction of PGC with asialoglycoprotein receptors (ASGPR) and cellular internalization in HepG2 cells was studied using in silico and uptake studies respectively. PGC was evaluated for its metal chelating, ferric ion reducing, superoxide, and lipid peroxide (LPO) inhibiting potential. Further, anticancer therapeutic potential of PGC was evaluated against N-nitrosodiethylamine (NDEA)-induced hepatocellular carcinoma in a mice model. After development of cancer, PGC was administered to the treatment group (0.5 mg/kg bw, intravenously), once a week for 4 weeks. Characterization studies of PGC revealed successful phosphorylation and galactosylation of chitosan. A strong interaction of PGC with ASGP-receptors was predicted by computational studies and cellular internalization studies demonstrated 98.76 ± 0.53% uptake of PGC in the HepG2 cells. A good metal chelating, ferric ion reducing, and free radical scavenging activity was demonstrated by PGC. The anticancer therapeutic potential of PGC was evident from the observation that PGC treatment increased number of tumor free animals (50%) (6/12) and significantly (p ≤ 0.05) lowered tumor multiplicity as compared to untreated tumor group.

Quinazolinone derivative BNUA-3 ameliorated [NDEA+2-AAF]-induced liver carcinogenesis in SD rats by modulating AhR-CYP1B1-Nrf2-Keap1 pathway.

Cytochrome P450 1B1, considered as one of the novel chemotherapeutic targets involved in cancer prevention and therapy is also associated with the conversion of procarcinogens into their active metabolites. The aryl hydrocarbon receptor (AhR) is responsible for mediating different biological responses to a wide variety of environmental pollutants and also causes transcriptional activation of cytochrome P450 enzymes including CYP1B1 and thus plays a pivotal role for initiating cancer and its progression. On the other hand, active carcinogenic metabolites and reactive oxygen species-mediated stress alter different molecular signalling pathways and gene expressions. Quinazoline derivatives are recognized for their diversified biological activities including anticancer properties. The current study was designed for evaluation of chemotherapeutic efficacy of a synthetic quinazolinone derivative BNUA-3 against hepatocellular cancer in Sprague-Dawley (SD) rats. A detailed in vivo analysis was performed by administrating BNUA-3 (15, 30 mg/kg b.w. for 28 days, i.p.) in N-Nitrosodiethylamine + 2-Acetylaminofluorene induced partially hepatectomized liver cancer in SD rats. This was followed by morphological evaluations, biochemical estimations and analysis of different mRNA and protein expressions. The results demonstrated the potency of BNUA-3 in efficient restoration of the altered morphology of liver, its protective effect against lipid peroxidation, enzymic and non-enzymic antioxidants levels in liver tissue which was disrupted after cancer induction. The study also demonstrated downregulation of AhR, CYP1B1 and Keap1 expressions with subsequent augmentation of protective Nrf2, HO-1, NQO1 and GSTA1 expressions thus, revealing the chemotherapeutic potency of BNUA-3 in inhibiting liver carcinogenesis through AhR/CYP1B1/Nrf2/Keap1 pathway.

Umbelliferone and esculetin protect against N-nitrosodiethylamine-induced hepatotoxicity in rats.

N-nitrosodiethylamine (NDEA), a nitrosamine compound, is known to cause liver damage through the generation of reactive oxygen species (ROS), resulting in oxidative damage to macromolecules such as DNA, and the consequent development of cancer. The present study examines the protective effects of two antioxidant coumarin compounds umbelliferone (Umb) and esculetin (Esc) against NDEA-induced hepatotoxicity when administered in the diet to male Wistar rats. The results show that treatment with Umb (0.5% w/w) and Esc (0.5% w/w) in the diet for 7 days significantly attenuates NDEA-induced liver damage, lowering serum alanine transaminase (ALT) levels, decreasing hepatic lipid peroxidation, and restoring total glutathione levels. To investigate the mechanism for the observed protective effect, the levels of the key protective enzymes NAD(P)H: quinone oxidoreductase 1 (NQO1), heme oxygenase (HO1), and glutathione S-transferase Pi (GSTP1) were measured by Western blotting following Umb and Esc administration. The results showed that Umb and Esc administration significantly increased the expression of NQO1 by 3.6- and 2.7-fold, HO1 by 2.7- and 3.2-fold, and GSTP1 by 2.8- and 3.2-fold, respectively. In conclusion, Umb and Esc are capable of protecting liver from NDEA-induced hepatotoxicity, and this is associated with the induction of protective enzymes.

Feasibility of NDEA formation control from DEDTC in chlorination/chloramination by pre-ozonation: Mechanisms and influencing factors.

N-nitrosodiethylamine (NDEA), which is the most toxic nitrosamine among the 9 detected species, has been widely detected in drinking water. Amines containing diethylamine (DEA) groups in the structure would generate NDEA during the disinfection processes. The aim of this study was to evaluate the feasibility of reducing NDEA formation from a commonly used dithiocarbamate pesticide sodium diethyldithiocarbamate (DEDTC) in subsequent chlorination and chloramination by pre-ozonation. The results demonstrated that NDEA could be generated directly during ozonation, its amounts increased from 0 to 14.34 µg/L with increasing ozone dosages (0-4 mg/L), which was higher than that chlorination (2.68 µg/L) and chloramination (4.91 µg/L) when the initial concentration of DEDTC was 20 µM. Pre-ozonation significantly raised NDEA formation from 2.68 to15.32 µg/L in subsequent chlorination; and that from 4.91 to 9.54 µg/L during subsequent chloramination processes. The addition of •OH scavenger tert-butanol (tBA) increased the production of NDEA from 8.14 to 20.80 µg/L during ozonation, and that from 6.76 to17.98 µg/L in O3/HClO process, 8.74 to 17.33 µg/L in O3/NH2Cl process. Except for NO3- and CO32-, most of the co-existing substances promoted NDEA generation from DEDTC under disinfection conditions. Based on the results of Gaussian theory calculations, GC/MS and UPLC-Q-TOFMS analysis, the influencing mechanisms of pre-ozonation on NDEA generation in the subsequent disinfection process were proposed. In addition, not only acute/chronic toxicity calculation but also luminescent bacteria test was performed to assess the possibility of pre-ozonation on the risk control of DEDTC. The research results fill a gap in the control of NDEA pollution and help to develop a safer ozone oxidation technology.

Anticancer therapeutic potential of multimodal targeting agent- "phosphorylated galactosylated chitosan coated magnetic nanoparticles" against N-nitrosodiethylamine-induced hepatocellular carcinoma.

Superparamagnetic iron oxide nanoparticles (SPIONs) are extensively used as carriers in targeted drug delivery and has several advantages in the field of magnetic hyperthermia, chemodynamic therapy and magnet assisted radionuclide therapy. The characteristics of SPIONs can be tailored to deliver drugs into tumor via "passive targeting" and they can also be coated with tissue-specific agents to enhance tumor uptake via "active targeting". In our earlier studies, we developed HCC specific targeting agent- "phosphorylated galactosylated chitosan"(PGC) for targeting asialoglycoprotein receptors. Considering their encouraging results, in this study we developed a multifunctional targeting system- "phosphorylated galactosylated chitosan-coated magnetic nanoparticles"(PGCMNPs) for targeting HCC. PGCMNPs were synthesized by co-precipitation method and characterized by DLS, XRD, TEM, VSM, elemental analysis and FT-IR spectroscopy. PGCMNPs were evaluated for in vitro antioxidant properties, uptake in HepG2 cells, biodistribution, in vivo toxicity and were also evaluated for anticancer therapeutic potential against NDEA-induced HCC in mice model in terms of tumor status, electrical properties, antioxidant defense status and apoptosis. The characterization studies confirmed successful formation of PGCMNPs with superparamagnetic properties. The internalization studies demonstrated (99-100)% uptake of PGCMNPs in HepG2 cells. These results were also supported by biodistribution studies in which increased iron content (296%) was noted inside the hepatocytes. Further, PGCMNPs exhibited no in vivo toxicity. The anticancer therapeutic potential was evident from observation that PGCMNPs treatment decreased tumor bearing animals (41.6%) and significantly (p ≤ 0.05) lowered tumor multiplicity. Overall, this study indicated that PGCMNPs with improved properties are efficiently taken-up by hepatoma cells and has therapeutic potential against HCC. Further, this agent can be tagged with 32P and hence can offer multimodal cancer treatment options via radiation ablation as well as magnetic hyperthermia.

Volatile N-nitrosamines in Spanish commercial meat products and in fermented sausages prepared with different ingoing amounts of nitrate and nitrite.

Spanish commercial dry fermented sausages and dry hams, manufactured with and without nitrate and/or nitrite have been screened for volatile N-nitrosamine (VNA) content. VNAs have been also quantified in experimental fermented sausages prepared with known ingoing amounts of curing salts. Solid phase microextraction followed by tandem quadrupole gas chromatography/mass spectrometry (GC-QQQ-MS) analysis allowed the identification and quantification of 8 VNAs, 5 of which were detected in the samples. The highest concentration of VNAs found in the commercial products was 5.4 µg/kg. The most frequently detected VNAs were N-nitrosodiphenylamine and N-nitrosopyrrolidine. Principal component analysis and cluster analysis did not show correlation between the content of VNAs and the use of nitrate/nitrite in the formula. In the experimental sausages N-nitrosodiphenylamine and N-nitrosopyrrolidine were only detected (0.55 µg/kg total concentration) when 150 mg/kg of both nitrate and nitrite were added to the formula without any antioxidant. The levels of VNAs detected in this study are similar to those reported in the literature in different fermented meat products and dry hams.

Metformin drugs under simulated gastric conditions can generate high nitrite-dependent levels of N-nitrosodimethylamine.

N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA), group 2A carcinogens, were detected in finished drug products, including metformin, ranitidine, sartans and other drugs which caused multiple recalls in the USA and Europe. Important studies also reported the formation of NDMA when ranitidine and nitrite were added to simulated gastric fluid. Our objective was to screen finished drug products from Europe and USA for nitrosamine impurities and investigate the formation of NDMA in metformin finished drug products when added to simulated gastric fluid. One dosage unit of 30 different commercially available drugs, including metformin, sartans, and ranitidine were tested for NDMA, NDEA, and dimethylformamide (DMF) impurities, using a liquid chromatography-mass spectrometry (LC-MS) method. Then, 6 metformin finished drug products were tested in stomach conditions for 2 h at 37 °C in a 100 mL solution with a pH of 2.5 and different nitrite concentrations (40, 10, 1, 0.1 mM) and tested for NDMA, and DMF using LC-MS. We measured NDMA, NDEA, and DMF in 30 finished drug products. NDMA and DMF were quantified for metformin drug products in simulated gastric fluid with different nitrite concentrations. None of the 30 drugs showed concerning levels of NDMA, NDEA, or DMF when tested as single tablets. However, when metformin tablets are added to simulated gastric fluid solutions with high nitrite concentrations (40 mM and 10 mM), NDMA can reach amounts of thousands of nanograms per tablet. At the closest concentration to physiologic conditions we used, 1 mM, NDMA is still present in the hundreds of nanograms in some metformin products. In this in vitro study, nitrite concentration had a very important effect on NDMA quantification in metformin tablets added to simulated gastric fluid. 1 mM nitrite caused an increase above the acceptable daily intake set by the U.S. Food and Drug Administration (FDA) for some of the metformin drugs. 10 mM, 40 mM nitrite solutions generated NDMA amounts exceeding by more than a hundred times the acceptable daily intake set by the FDA of 96 nanograms. These findings suggest that metformin can react with nitrite in gastric-like conditions and generate NDMA. Thus, patients taking metformin could be exposed to NDMA when high nitrite levels are present in their stomach, and we recommend including a statement within the Patient Package Inserts/Instructions for use.

Investigation of Carcinogenic Impurities of N-Nitrosamines in Sartan Pharmaceutical Products Marketed in Brazil: Development and Validation of Method Based on High-Performance Liquid Chromatography-Tandem Mass Spectrometry.

N-nitrosamines (NA) impurities have unexpectedly been found in sartan products, angiotensin II receptor antagonists that are used to control hypertension, representing an urgent concern for industry, global regulators and for the patients. In this study, an HPLC-MS/MS method was developed and validated for the quantification of six NA (N-nitrosodimethylamine, N-Nitroso-N-methyl-4-aminobutyric acid, N-Nitrosodiethylamine, N-ethyl-N-nitroso-2-propanamine, N-nitroso-diisopropylamine and N-nitroso-di-n-butylamine) in losartan, valsartan, olmesartan, irbesartan, candesartan and telmisartan products. The method was validated in terms of sensitivity, linearity, accuracy, precision, robustness and stability. The limits of quantification were 100, 31.25, 250, 33, 312.5 and 125 µg kg-1 in losartan, valsartan, olmesartan, irbesartan, candesartan and telmisartan samples, respectively, which met the sensitivity requirements for the limits set by Food and Drug Administration of the United States. The standard curves showed good linearity. The recoveries ranged from 93.06 to 102.23% in losartan matrix, 83 to 85.9% in valsartan, 96.1 to 101.2% in olmesartan, 89.2 to 97.5% in irbesartan, 93.4 to 132.0% in candesartan and 62.3 to 106.2% in telmisartan matrix. The other parameters met the validation criteria, the good sensitivity and precision, high accuracy and simple and fast analysis provides a reliable method for quality control of NA in sartan pharmaceutical products. The developed method was successfully applied for the determination of N-nitrosamines in 71 sartan products marketed in Brazil.

In silico and in vivo anti-angiogenic validation on ethanolic extract of Curcuma longa and curcumin compound in hepatocellular carcinoma through mitogen activated protein kinase expression in male and female wistar rats.

Hepatocellular carcinoma (HCC) is the most frequent primary malignancy of the liver. The aim of this study is to evaluate the comparative in silico and in vivo ameliorative potential of the ethanolic extract of Curcuma longa (EECL) in male and female Wistar rats administered N-nitrosodiethylamine-induced hepatocellular carcinoma. The MAPK compound was obtained from a protein data bank (PDB ID: 7AUV) for molecular docking. One hundred and twenty Wistar rats, were randomly selected into twelve groups (n = 5): Group A received regular diets as a basal control; groups B to G were administered 100 mg/kg NDEA twice in two weeks; while groups C to E received 200 mg/kg, 400 mg/kg, and 600 mg/kg of EECL; group F was treated with 200 mg/kg pure curcumin; and group G received 100 mg/kg Sylibon-140. Group H received only 200 mg/kg pure curcumin, and group I received 200 mg/kg of dimethylsulfoxide (DMSO). Groups J, K, and L received 200 mg/kg, 400 mg/kg and 600 mg/kg of EECL. MAPK and AFP mRNA in Wistar rats administered NDEA were upregulated as compared to EECL groups. In conclusion, the in silico and in vitro study validates the mitigating role of ethanolic extract of Curcuma longa and pure curcumin.

Involvement of PI3K/HIF-1α/c-MYC/iNOS Pathway in the Anticancer Effect of Suaeda vermiculata in Rats.

Suaeda vermiculata Forssk. ex JF Gmel. (SV), a traditional known plant, has shown in vitro cytotoxic activity against HepG2 and HepG-2/ADR (doxorubicin-resistant cells) liver cell carcinoma cell lines, as well as hepatoprotection against paracetamol and carbon tetrachloride (CCl4)-induced liver injury. The current study evaluated the protective effect of SV, administered against N-diethylnitrosamine (NDEA)-induced HCC in rats. The possible modulatory effect of SV on the PI3K/HIF-1α/c-MYC/iNOS pathway was investigated. Sixty male adult albino rats (200 ± 10 g) were equally classified into five groups. Group I served as a control; Group 2 (SV control group) received SV (p.o., 200 mg/kg body weight); Group 3 (NDEA-administered rats) received freshly prepared NDEA solution (100 mg/L); and Groups 4 and 5 received simultaneously, for 16 weeks, NDEA + SV extract (100 and 200 mg/kg, orally). NDEA-treated rats displayed significant increases in serum levels of AFP, CEA, PI3K, malondialdehyde (MDA), epidermal growth factor receptor (EGFR), and vascular endothelial growth factor (VEGFR), with increased liver tissue protein expression of fibrinogen concomitant and significantly decreased concentrations of antioxidant parameters (catalase (CAT), superoxide dismutase (SOD), and reduced glutathione (GSH)) in comparison to normal rats. On the flip side, AFP, CEA, PI3K, MDA, EGFR, and VEGFR serum levels were significantly reduced in rats that received NDEA with SV, both at low (SV LD) and high (SV HD) doses, accompanied by significant improvements in antioxidant parameters compared to the NDEA-treated group. Conclusions: SV possesses a significant hepatoprotective effect against NDEA-induced HCC via inhibiting the PI3K/HIF-1α/c-MYC/iNOS pathway, suggesting that SV could be a promising hepatocellular carcinoma treatment.

Using network data envelopment analysis to assess the sustainability and resilience of healthcare supply chains in response to the COVID-19 pandemic.

The widespread outbreak of a new Coronavirus (COVID-19) strain has reminded the world of the destructive effects of pandemic and epidemic diseases. Pandemic outbreaks such as COVID-19 are considered a type of risk to supply chains (SCs) affecting SC performance. Healthcare SC performance can be assessed using advanced Management Science (MS) and Operations Research (OR) approaches to improve the efficiency of existing healthcare systems when confronted by pandemic outbreaks such as COVID-19 and Influenza. This paper intends to develop a novel network range directional measure (RDM) approach for evaluating the sustainability and resilience of healthcare SCs in response to the COVID-19 pandemic outbreak. First, we propose a non-radial network RDM method in the presence of negative data. Then, the model is extended to deal with the different types of data such as ratio, integer, undesirable, and zero in efficiency measurement of sustainable and resilient healthcare SCs. To mitigate conditions of uncertainty in performance evaluation results, we use chance-constrained programming (CCP) for the developed model. The proposed approach suggests how to improve the efficiency of healthcare SCs. We present a case study, along with managerial implications, demonstrating the applicability and usefulness of the proposed model. The results show how well our proposed model can assess the sustainability and resilience of healthcare supply chains in the presence of dissimilar types of data and how, under different conditions, the efficiency of decision-making units (DMUs) changes.

Regulatory Updates and Analytical Methodologies for Nitrosamine Impurities Detection in Sartans, Ranitidine, Nizatidine, and Metformin along with Sample Preparation Techniques.

Nitrosamine impurities have been detected in various pharmaceutical products in recent days. Various sartans, ranitidine, nizatidine, and metformin have been recalled from the markets due to the high limit of nitrosamine impurities. This review aims to provide a brief overview of nitrosamine impurities, detection methods in detail, mechanism of action of nitrosamine impurities, sample preparation techniques, and regulatory limits. Numerous reported nitrosamine impurities also have been discussed with chemical structure. Various detection methods including LC-MS/MS, GC-MS-HS, and HPLC for nitrosamine impurities along with sartans, ranitidine, nizatidine, and metformin are being discussed in this review article. Various sample preparation techniques such as solid-phase extraction, liquid-liquid extraction, and rapid-fire techniques have also been discussed. This review will provide the detail information to the analytical manpower working in various quality control laboratories as well as in research organizations. HighlightsDetection of nitrosamine (NA) impurities in drug substances as well as finished products.HPLC, LC-MS/MS, and GC-MS/HS/AS discussed for the quantificationSolid-phase extraction, liquid-liquid extraction, and rapid-fire method for NA sample preparationMechanistic approach for nitrosamine formation and its removal strategiesRegulatory limits for NA impurities incorporated.

The Effect of Tobacco Smoke N-Nitrosamines, NNK and NDEA, and Nicotine, on DNA Mismatch Repair Mechanism and miRNA Markers, in Hypopharyngeal Squamous Cell Carcinoma: An In Vivo Model and Clinical Evidence.

Deregulation of the DNA mismatch repair (MMR) mechanism has been linked to poor prognosis of upper aerodigestive tract cancers. Our recent in vitro data have provided evidence of crosstalk between deregulated miRNAs and MMR genes, caused by tobacco smoke (TS) N-Nitrosamines, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), in hypopharyngeal cells. Here, we explored whether chronic exposure to TS components can affect MMR mechanism and miRNA profiles in hypopharyngeal mucosa. Using a mouse model (C57Bl/6J wild type) of in vivo 14-week exposure to NNK (0.2 mmol/L) and N-Nitrosodiethylamine (NDEA; 0.004 mmol/L), with or without nicotine (0.02 µmol/L), we provide direct evidence that TS components can promote dysplasia, significant downregulation of Msh2 and Mlh1 genes and deregulation of miR-21, miR-155, miR-34a, and miR-451a. By analyzing eight human specimens from tobacco smokers and eight controls, we provide clinical evidence of a significant reduction in hMSH2 and hMLH1 mRNAs in hypopharyngeal squamous cell carcinoma (HSCC). In summary, deregulation of the MMR mechanism and miRNAs is caused by chronic exposure to TS-related N-Nitrosamines, with or without nicotine, in the early stages of upper aerodigestive tract carcinogenesis, and can also be detected in human HSCC. Thus, we encourage future studies to further elucidate a possible in vivo dose-dependent effect of individual or combined N-Nitrosamines, NNK and/or NDEA, and nicotine, on the MMR mechanism and their clinical testing to elaborate prognosis and risk assessment.

Presence of nitrosamine impurities in medicinal products.

In 2018, some sartan medicinal products were reported to be contaminated with nitrosamine compounds, which are potent mutagenic carcinogens. Two nitrosamines received particular attention: N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA). These have since been confirmed in different types of medicinal products, including ranitidine and metformin. Consequently, the European Medicines Agency (EMA) started an investigation into the cause of contamination and an assessment of the risk to patients taking contaminated medicinal products. The main source of contamination were changes in production, which involves combinations of amines and nitrogen compounds and the use of specific catalysts and reagents. Withdrawals of medicinal products that took place in Croatia did not lead to a shortage of sartan- or metformin-containing medicines. Moreover, ranitidine had been preventively withdrawn all over the EU, including Croatia, creating shortages at the time, but was subsequently replaced with therapeutic alternatives.

Mitochondria-targeted antioxidant, mito-TEMPO mitigates initiation phase of N-Nitrosodiethylamine-induced hepatocarcinogenesis.

Targeting mitochondrial oxidative stress during initial stages of hepatocarcinogenesis can be an effective and promising strategy to prevent hepatocellular carcinoma (HCC). In the present study, mitochondria targeted antioxidant, mito-TEMPO was administered to male BALB/c mice at a dosage 0.1 mg/kg b.w. (intraperitoneal) twice a week, followed by single N-Nitrosodiethylamine (NDEA) intraperitoneal injection (10 mg/kg b.w.). After 24 h of NDEA administration, animals were sacrificed, blood and liver tissue were collected. Liver injury markers, histoarchitecture, antioxidant defence status, mitochondrial reactive oxygen species (ROS), lipid peroxidation (LPO), mitochondrial dysfunction analysis, and mitochondrial membrane potential were investigated. Mito-TEMPO pre-treatment protected animals from the damaging effects of NDEA as observed by normalization of liver injury markers. NDEA metabolism resulted in a significantly increased intracellular and mitochondrial ROS generation with concomitant increase in LPO formation. The activity of mitochondrial complex I, complex II, malate dehydrogenase were significantly reduced and mitochondrial membrane potential was increased. Mito-TEMPO effectively scavenged NDEA-induced ROS generation and reduced LPO formation. A significant improvement was also observed in the activity of mitochondrial complex I, complex II, malate dehydrogenase and normalisation of mitochondrial membrane potential. Results suggested that mito-TEMPO had significant impact on the initiation phase of hepatocarcinogensis which could be one of the reason for its reported chemopreventive effect.

Intake of volatile nitrosamines by Chinese residents in different provinces via food and drinking water.

N-nitrosamines are potent carcinogens, particularly N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA), which are commonly found in a variety of foods and drinking water. We calculated the food and drinking water intakes of NDMA, NDEA, and total volatile nitrosamines (TVNA) by Chinese residents in different provinces by multiplying the reported total diet study results by the nitrosamine contents in food and drinking water. The weighted content of nitrosamines in each category of foods and the concentration of nitrosamines in drinking water was obtained through literature review. The exogenous NDMA, NDEA and TVNA intakes of adult residents in 20 provinces ranged from 171 to 425 ng/d, 41 to 140 ng/d and 373 to 1028 ng/d, respectively. The main contributors to NDMA and TVNA intakes were vegetables, cereals, aquatic products, and meats while the main sources of NDEA intake were vegetables and cereals. The average total NDMA intake per capita in China (251 ng/d) was similar to that in Germany in 1991 (231 ng/d) but higher than that in the United States (136 ng/d), Canada (87.6 ng/d) and France (188 ng/d). Large differences in nitrosamine intakes were observed between the coastal provinces and inland provinces. Drinking water was estimated to contribute 13.1%, 1.3% and 10.8% of the exogenous intakes of NDMA, NDEA and TVNA, respectively. Based on our results, we recommend setting the NDMA drinking water criterion of 40 ng/L. Overall, this study presents basic information regarding nitrosamines intake via food and drinking water in China that will facilitate risk assessment, generation of health advisories and policy making.