Subcellular distribution and Nrf2/Keap1-interacting properties of Glutathione S-transferase P in hepatocellular carcinoma.

The oncogene and drug metabolism enzyme glutathione S-transferase P (GSTP) is also a GSH-dependent chaperone of signal transduction and transcriptional proteins with key role in liver carcinogenesis. In this study, we explored this role of GSTP in hepatocellular carcinoma (HCC) investigating the possible interaction of this protein with one of its transcription factor and metronome of the cancer cell redox, namely the nuclear factor erythroid 2-related factor 2 (Nrf2). Expression, cellular distribution, and function as glutathionylation factor of GSTP1-1 isoform were investigated in the mouse model of N-nitrosodiethylamine (DEN)-induced HCC and in vitro in human HCC cell lines. The physical and functional interaction of GSTP protein with Nrf2 and Keap1 were investigated by immunoprecipitation and gene manipulation experiments. GSTP protein increased its liver expression, enzymatic activity and nuclear levels during DEN-induced tumor development in mice; protein glutathionylation (PSSG) was increased in the tumor masses. Higher levels and a preferential nuclear localization of GSTP protein were also observed in HepG2 and Huh-7 hepatocarcinoma cells compared to HepaRG non-cancerous cells, along with increased basal and Ebselen-stimulated levels of free GSH and PSSG. GSTP activity inhibition with the GSH analogue EZT induced apoptotic cell death in HCC cells. Hepatic Nrf2 and c-Jun, two transcription factors involved in GSTP expression and GSH biosynthesis, were induced in DEN-HCC compared to control animals; the Nrf2 inhibitory proteins Keap1 and ß-TrCP also increased and oligomeric forms of GSTP co-immunoprecipitated with both Nrf2 and Keap1. Nrf2 nuclear translocation and ß-TrCP expression also increased in HCC cells, and GSTP transfection in HepaRG cells induced Nrf2 activation. In conclusion, GSTP expression and subcellular distribution are modified in HCC cells and apparently contribute to the GSH-dependent reprogramming of the cellular redox in this type of cancer directly influencing the transcriptional system Nrf2/Keap1.

A systematic review on multipotent carcinogenic agent, N-nitrosodiethylamine (NDEA), its major risk assessment, and precautions.

The International Agency for Research on Cancer has classified N-nitrosodiethylamine (NDEA) as a possible carcinogen and mutagenic substances, placing it in category 2A of compounds that are probably harmful to humans. It is found in nature and tobacco smoke, along with its precursors, and is also synthesized endogenously in the human body. The oral or parenteral administration of a minimal quantity of NDEA results in severe liver and kidney organ damage. The NDEA required bioactivation by CYP450 enzyme to form DNA adduct in the alkylation mechanism. Thus, this bioactivation directs oxidative stress and injury to cells due to the higher formation of reactive oxygen species and alters antioxidant system in tissues, whereas free radical scavengers guard the membranes from NDEA-directed injury in many enzymes. This might be one of the reasons in the etiology of cancer that is not limited to a certain target organ but can affect various organs and organ systems. Although there are various possible approaches for the treatment of NDEA-induced cancer, their therapeutic outcomes are still very dismal. However, several precautions were considered to be taken during handling or working with NDEA, as it considered being the best way to lower down the occurrence of NDEA-directed cancers. The present review was designed to enlighten the general guidelines for working with NDEA, possible mechanism, to alter the antioxidant line to cause malignancy in different parts of animal body along with its protective agents. Thus, revelation to constant, unpredictable stress situations even in common life may remarkably augment the toxic potential through the rise in the oxidative stress and damage of DNA.

Modulatory mechanisms of copperII-albumin complex toward N-nitrosodiethylamine-induced neurotoxicity in mice via regulating oxidative damage, inflammatory, and apoptotic signaling pathways.

N-nitrosodiethylamine (ND) is an extremely toxic unavoidable environmental contaminant. CopperII-albumin (CuAB) complex, a newly developed Cu complex, showed antioxidant and anti-inflammatory potential. Hereby, we explored the plausible neuroprotective role of CuAB complex toward ND-evoked neurotoxicity in mice. Twenty-four male mice were sorted into 4 groups (6 mice each). Control group, mice were administered oral distilled water; and CuAB group, mice received CuAB complex at a dose of 817 µg/kg orally, three times weekly. In ND group, ND was given intraperitoneally (50 mg/kg body weight, once weekly for 6 w). CuAB+ND group, mice were administered a combination of CuAB and ND. The brain was quickly extracted upon completion of the experimental protocol for the evaluation of the oxidative/antioxidative markers, inflammatory cytokines, and histopathological examination. Oxidative stress was induced after ND exposure indicated by a reduction in GSH and SOD1 level, with increased MDA level. In addition, decreased expression of SOD1 proteins, Nrf2, and 5-HT mRNA expression levels were noticed. An apoptotic cascade has also been elicited, evidenced by overexpression of Cyt c, Cl. Casp 3. In addition, increased regulation of proinflammatory genes (TNF-α, IL-6, iNOS, Casp1, and NF-κB (p65/p50); besides, increment of protein expression of P-IKBα and reduced expression of IKBα. Pretreatment with CuAB complex significantly ameliorated ND neuronal damage. Our results recommend CuAB complex supplementation because it exerts neuroprotective effects against ND-induced toxicity.

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.

Biomimetic synthesis of silver nanoparticles for treatment of N-Nitrosodiethylamine-induced hepatotoxicity.

The development of bioengineered nanoparticles has attracted considerable universal attention in the field of medical science and disease treatment. Current studies were executed to evaluate the hepatoprotective activity of biosynthesized silver nanoparticles (AgNPs). Their characterization was performed by UV-Visible analysis, fourier transform infrared spectroscopy, transmission electron microscopy (TEM), scanning electron microscope (SEM), and Zeta analyses. In in vivo studies, albino rats (180 ± 10 g) were persuaded with model hepatic toxicant N-nitrosodiethylamine (NDEA) and subsequently cotreated with Morus multicaulis at 100 mg/kg and AgNPs at 100 µg/kg dose. NDEA administration elevates the levels of liver function test biomarkers, which were reinstated to normal by cotreatment of test drugs. The oxidative stress and concentration of drug-metabolizing enzyme increase after induction of toxicant (NDEA), these markers are restored toward normal after cotreatment of nano-drug. Treatments of M. multicaulis extract did not show such significant protection. The NDEA-treated groups showed a significant rise in the level of cytokines (interleukin [IL-6] and IL-10) and reached normal with subsequent treatment with AgNPs. Histopathological studies also exhibited the curative effect of AgNPs in the same manner. Thus current results strongly suggest that biomimetic AgNPs could be used as an effective drug against hepatic alteration.

Citral inhibits N-nitrosodiethylamine-induced hepatocellular carcinoma via modulation of antioxidants and xenobiotic-metabolizing enzymes.

Hepatocellular carcinoma (HCC) ranks the sixth position among various cancers worldwide. Recent research shows that natural and dietary compounds possess many therapeutic effects. Citral is a monoterpene aldehyde that contains geranial and neral. The present study was considered to study the role of citral against N-nitrosodiethylamine (NDEA)-induced HCC via modulation of antioxidants and xenobiotic-metabolizing enzymes in vivo. NDEA-alone-administered group II animals profoundly showed increased tumor incidence, reactive oxygen species, liver marker enzyme levels, serum bilirubin levels, tumor markers of carcinoembryonic antigen, α-fetoprotein, proliferative markers of argyrophilic nucleolar organizing regions, proliferating cell nuclear antigen (PCNA) expressions, phase I xenobiotic-metabolic enzymes and simultaneously decreased antioxidants, and phase II enzymes levels. Citral (100 mg/kg b.w.) treatment significantly reverted the levels in group III cancer-bearing animals when compared to group II cancer-bearing animals. In group IV animals, citral-alone administration did not produce any adverse effect during the experimental condition. Based on the results, citral significantly inhibits the hepatocellular carcinogenesis through restoring the antioxidants and phase II xenobiotic-enzyme levels; thereby, it strongly proves as an antiproliferative agent against rat HCC.

Lesson Learnt from Recall of Valsartan and Other Angiotensin II Receptor Blocker Drugs Containing NDMA and NDEA Impurities.

The presence of N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) impurities in angiotensin II receptor blocker (ARB) drugs containing tetrazole ring has triggered worldwide product recalls. The purpose of this article is to identify the potential gap area in current pharmaceutical industry practice that might have led to the NMDA and NDEA impurities escaping the drug manufacturer's and FDA's attention. The impact of process change was not adequately assessed by the manufacturer of contaminated APIs (active pharmaceutical ingredients), and potential for generation of mutagenic or other toxic impurities was not considered. The safety and risk associated with a chemical synthetic process was also not evaluated. This is primarily due to current industry practice which focuses on controlling the impurities above reporting threshold. ICH Q3A and FDA guidance on genotoxic and carcinogenic impurities in drug substances and products need to be integrated so that the ICH Q3A decision tree (attachment 3) begins by checking whether the synthetic process has been evaluated for the potential to generate toxic impurities. The compliance with ICH Q3A limits should be carried out only after the process has been determined to be safe without the risk of generating mutagenic and carcinogenic impurities.

[Control of highly toxic N-nitrosamines (N-nitrosodimethylamine and N-nitrosodiethylamine) content in baby's cereals].

This paper proposes gas chromatography-mass-spectrometry method for determination of N-nitrosamines (N-nitrosodimethylamine and N-nitrosodiethylamine) in dry baby cereals (milk and milk-free). According to the results of the experimental studies, the method of sample preparation has been substantiated. This is the method of distillation with superheated steam, concentration of distillate on cartridges of automatic system of solid phase extraction. Optimal conditions for chromatography-mass spectrometry analysis has been selected (GC/MS). Analysis of the cereals (milk and milkfree) on the content of N-nitrosodimethylamine and N-nitrosodiethylamine in concentration range 0.0055-0.0109 mg/kg allowed to determine high content of the determined components by the sum of N-nitrosamines in cereals' samples of different manufacturers: oatmeal with milk, buckwheat with milk, oatmeal with milk and banana, milk-free maize cereal. In the multigrain milk cereal and buckwheat with milk, peaches and apricots the content N-nitrosamines was not detected (0.0004-0.00066 mg/kg). To confirm the presence of N-nitrosamines identified in a sample of oatmeal with milk, the identification in SCAN mode has been performed. Mass-spectrum of N-nitrosodimethylamine and N-nitrosodiethylamine in examined samples were compared with massspectrums that were included in library bank of mass-spectral data NIST 08.L.

Protective Effects of Total Glucosides of Paeony on N-nitrosodiethylamine-induced Hepatocellular Carcinoma in Rats via Down-regulation of Regulatory B Cells.

Total glucoside of paeony (TGP), extracted from the root of Paeonia Lactiflora, has been known to show anti-inflammatory, anti-oxidative, hepato-protective and immuno-regulatory activities. The aim of this present study was to determine the anti-tumor effect of TGP against N-nitrosodiethylamine (DEN)-induced hepatocellular carcinoma (HCC) in rats, and to find the related mechanisms. Rat HCC model was established by intragastrically administrating with DEN (8 mg/kg). We found the number of tumor nodules and the index of liver and spleen were increased in the model group compared with the normal group, and was significantly decreased by TGP. Additionally, TGP obviously improved the hepatic pathological lesions induced by DEN, and decreased the elevated levels of serum alanine aminotransferase (ALT), glutamic oxalacetic transaminase (AST), alkaline phosphatase (ALP) and alpha fetoprotein (AFP) by DEN. Moreover, TGP decreased the level of B cell-activating factor (BAFF) and the proportion of IL-10-producing regulatory B cells (Bregs), and the decrease of BAFF by TGP is positively correlated to the decrease of IL-10-producing Bregs by TGP. These results suggest that TGP had a good therapeutic action on DEN-induced HCC rats, which might be due to its down-regulation of Bregs through reducing the level of BAFF.

Protective Assessment of Euphorbia neriifolia and its Isolated Flavonoid Against N-nitrosodiethylamine-induced Hepatic Carcinogenesis in Male Mice: A Histopathological Analysis.

AIMS: The aim of this study was to examine the impacts of N-nitrosodiethylamine (DENA), a potent environment carcinogen on liver tissue of mice which was attenuated by isolated flavonoid and hydro-ethanolic extract of Euphorbia neriifolia (HEEN) leaves. MATERIALS AND METHODS: Carcinogenicity was induced in albino mice by a single oral administration of DENA (50 mg/kg body weight). The HEEN (150 and 400 mg/kg body weight), butylated hydroxyanisole (BHA; 0.5 and 1%) and E. neriifolia flavonoid (ENF; 50 mg/kg body weight) were estimated to examine the possible anti-cancer potential. RESULTS: DENA exposed animals showed alterations in normal hepatic histo-architecture, which comprised of necrosis (N), dilated sinusoids and vacuolization of the cells. Mice treated with E. neriifolia lower (ENL) and higher (ENH) dose and ENF before intoxicated with DENA showed that the liver cells were normal, with very little necrosis (Day 31). On the other hand, BHA higher (BHAH) and lower (BHAL) dose failed to diminish the abnormalities caused by the DENA. CONCLUSION: Results of the present study suggests that the ENH and ENF protects the hepatic tissue against DENA-induced hepatic carcinoma. The results could also be expressed in the order of ENH> ENF> ENL> BHAH> BHAL.

Compromised very-low density lipoprotein induced polyunsaturated triglyceride accumulation in N-nitrosodiethylamine-induced hepatic steatosis.

N-Nitrosodiethylamine (NDEA), a carcinogen in some foods and medications, is linked to liver damage similar to non-alcoholic fatty liver disease (NAFLD). This study explores how NDEA disrupts liver lipid metabolism. Sprague-Dawley rats were given two doses of NDEA (100 mg/kg) orally, 24 h apart. Liver response was assessed through tissue staining, blood tests, and biochemical markers, including fatty acids, lipid peroxidation, and serum very-low density lipoprotein (VLDL) levels. Additionally, lipidomic analysis of liver tissues and serum was performed. The results indicated significant hepatic steatosis (fat accumulation in the liver) following NDEA exposure. Blood analysis showed signs of inflammation and liver damage. Biochemical tests revealed decreased liver protein synthesis and specific enzyme alterations, suggesting liver cell injury but maintaining mitochondrial function. Increased fatty acid levels without a rise in lipid peroxidation were observed, indicating fat accumulation. Lipidomic analysis showed increased polyunsaturated triglycerides in the liver and decreased serum VLDL, implicating impaired VLDL transport in liver dysfunction. In conclusion, NDEA exposure disrupts liver lipid metabolism, primarily through the accumulation of polyunsaturated triglycerides and impaired fat transport. These findings provide insight into the mechanisms of NDEA-induced liver injury and its progression to hepatic steatosis.

An Agile and Accurate Approach for N-Nitrosamines Detection and Quantification in Medicines by DART-MS.

N-nitrosamines (NAs) are prevalent mutagenic impurities in various consumer products. Their discovery in valsartan-containing medicines in 2018 prompted global regulatory agencies to set guidelines on their presence and permissible levels in pharmaceuticals. In order to determine the NAs content in medicines, efficient and sensitive analytical methods have been developed based on mass spectrometry techniques. Direct analysis in real time-mass spectrometry (DART-MS) has emerged as a prominent ambient ionization technique for pharmaceutical analysis due to its high-throughput capability, simplicity, and minimal sample preparation requirements. Thus, in this study DART-MS was evaluated for the screening and quantification of NAs in medicines. DART-MS analyses were conducted in positive ion mode, for both direct tablet analysis and solution analysis. The analytical performance was evaluated regarding linearity, precision, accuracy, limits of detection, and quantification. The DART-MS proved to be suitable for the determination of NAs in medicines, whether through direct tablet analysis or solution analysis. The analytical performance demonstrated linearity in the range from 1.00 to 200.00 ng mL-1, limits of quantification about 1.00 ng mL-1, precision and accuracy lower than 15%, and no significant matrix effect for six drug-related NAs. In conclusion, the DART-MS technique demonstrated to be an alternative method to determine NAs in medicines, aligning with the principles of green chemistry.

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.

Chemopreventive Role of Euphorbia neriifolia (Linn) and its Isolated Flavonoid Against N-Nitrosodiethylamine-induced Renal Histopathological Damage in Male Mice.

AIMS: This study is an attempt to evaluate the tissue protective efficacy of isolated flavonoid and hydro-ethanolic extract of Euphorbia neriifolia (HEEN) leaves against N-nitrosodiethylamine (DENA) induced renal carcinoma. MATERIALS AND METHODS: Carcinogenicity was induced in Albino mice by oral administration of DENA (50 mg/kg body weight). The HEEN (150 and 400 mg/kg body weight), Butylated hydroxyanisole (BHA; 0.5 and 1%), and Euphorbia neriifolia flavonoid (ENF; 50 mg/kg body weight) were evaluated for their possible tissue carcinogenesis protective potential. RESULTS: DENA treated animals showed alterations in normal renal histo-architecture, which comprised of necrosis (N) and vacuolization of the cells. On the other hand, the mice treated with Euphorbia neriifolia lower (ENL) and higher (ENH) dose and ENF before intoxicated with DENA showed that the renal cells were normal (Day 31). Whereas, BHA higher (BHAH) and lower (BHAL) dose failed to diminish the abnormalities caused by DENA. CONCLUSIONS: The findings of the present study Suggested that ENH and ENF showed highest renal-protective activity among all the pretreatments. The results could also be expressed in the order of ENH > ENF > ENL > BHAH > BHAL.

Comparison of the transgenic rodent mutation assay, error corrected next generation duplex sequencing, and the alkaline comet assay to detect dose-related mutations following exposure to N-nitrosodiethylamine.

N-Nitrosodiethylamine (NDEA), a well-studied N-nitrosamine, was tested in rats to compare the dose-response relationship of three genotoxicity endpoints. Mutant / mutation frequencies were determined using the transgenic rodent (TGR) gene mutation assay and error corrected next generation sequencing (ecNGS) (i.e., duplex sequencing (DS)), and genetic damage was detected by the alkaline comet assay. Big Blue® (cII Locus) animals (n = 6 per dose group) were administered doses of 0.001, 0.01, 0.1, 1, 3 mg/kg/day NDEA by oral gavage. Samples were collected for cII mutation and DS analyses following 28-days of exposure and 3 days recovery. In a separate study, male Sprague-Dawley (SD) rats (n = 6 per dose group) were administered the same doses by oral gavage for two consecutive days and then samples collected for the alkaline comet assay. A dose-related increase in mutant / mutation frequencies of the liver but not duodenum was observed using the TGR assay and DS with DS resulting in a slightly more sensitive response, with a lower benchmark dose (BMD). In addition, a dose-related increase in percent tail DNA was observed in the liver using the alkaline comet assay. Therefore, DS and comet assays showed good utility for hazard identification and dose-response analysis of a representative N-nitrosamine comparable to the TGR gene mutation assay.

A survey of industrial N-nitrosamine discharges in Switzerland.

N-nitrosamines are formed during different industrial processes and are of significant concern due to their carcinogenic and mutagenic properties. This study reports concentrations of N-nitrosamines in eight different industrial wastewater treatment plants in Switzerland and the variability of their abundance. Only four N-nitrosamines species, N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosodibutylamine (NDPA) and N-nitrosomorpholine (NMOR) were above the limit of quantification in this campaign. Remarkably high concentrations (i.e. up to 975 µg NDMA/L, 90.7 µg NDEA/L, 1.6 µg NDPA/L and 710 µg NMOR/L) of these N-nitrosamines were detected at seven of eight sites. These concentrations are two to five orders of magnitude higher than those typically detected in municipal wastewater effluents. These results suggest that industrial effluents may be a major source of N-nitrosamines. Although very high concentrations of N-nitrosamine have been detected in industrial discharges, various processes in surface water can partially mitigate their concentrations (e.g. photolysis, biodegradation and volatilization) and hence the risk to human health and aquatic ecosystems. Nevertheless, there is little information on long-term effects on aquatic organisms and therefore the discharge of N-nitrosamines to the environment should be avoided until the impact on ecosystems is assessed. During winter a less efficient mitigation of N-nitrosamines can be expected (lower biological activity, less sunlight) and therefore, emphasis should be put on this season in future risk assessment studies.

Metformin Prevents NDEA-Induced Memory Impairments Associated with Attenuating Beta-Amyloid, Tumor Necrosis Factor-Alpha, and Interleukin-6 Levels in the Hippocampus of Rats.

N-nitrosodiethylamine (NDEA) is a potential carcinogen known to cause liver tumors and chronic inflammation, diabetes, cognitive problems, and signs like Alzheimer's disease (AD) in animals. This compound is classified as probably carcinogenic to humans. Usual sources of exposure include food, beer, tobacco, personal care products, water, and medications. AD is characterized by cognitive decline, amyloid-ß (Aß) deposit, tau hyperphosphorylation, and cell loss. This is accompanied by neuroinflammation, which involves release of microglial cytokines, such as tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin 1ß (IL-1ß), by nuclear factor kappa B (NF-κB) upregulation; each are linked to AD progression. Weak PI3K/Akt insulin-signaling inhibits IRS-1 phosphorylation, activates GSK3ß and promotes tau hyperphosphorylation. Metformin, an antihyperglycemic agent, has potent anti-inflammatory efficacy. It reduces proinflammatory cytokines such as IL-6, IL-1ß, and TNF-α via NF-κB inhibition. Metformin also reduces reactive oxidative species (ROS) and modulates cognitive disorders reported due to brain insulin resistance links. Our study examined how NDEA affects spatial memory in Wistar rats. We found that all NDEA doses tested impaired memory. The 80 µg/kg dose of NDEA increased levels of Aß1-42, TNF-α, and IL-6 in the hippocampus, which correlated with memory loss. Nonetheless, treatment with 100 mg/kg of metformin attenuated the levels of pro-inflammatory cytokines and Aß1-42, and enhanced memory. It suggests that metformin may protect against NDEA-triggered memory issues and brain inflammation.

Effect of Black Garlic on Microbiological Properties, Lipid Oxidation, Residual Nitrite, Nitrosamine Formation and Sensory Characteristics in a Semi-Dry Fermented Sausage.

This study was conducted with the aim of determining the effects of different black garlic (BG) levels (1%, 2% and 3%) on quality characteristics of a semi-dry fermented sausage (heat-treated sucuk). In addition, the effect of cooking time (0, 1 or 3 min at 180 °C on a hot plate) on nitrosamine formation was investigated. Fresh garlic (FG, 1%) was evaluated as the control group. BG (2% and 3%) caused a reduction in the count of lactic acid bacteria while leading to an increase in pH. FG1% gave the highest number of Micrococcus/Staphylococcus, as well as aw value. The thiobarbituric acid reactive substance (TBARS) value increased with increasing BG levels. FG (1%) showed the highest residual nitrite amount (p < 0.05). The scores for color, taste and general acceptability were reduced by the use of BG (p < 0.05). No significant difference was observed between the garlic treatments in terms of N-Nitrosodimethylamine (NDMA) and N-Nitrosodiethylamine (NDEA) when no additional cooking was applied. Cooking time was determined to have no significant effect on NDMA in 3% BG. The use of BG caused an increase in N-Nitrosopiperidine (NPIP) (p < 0.05). As for PCA, a closer correlation between NPIP and the groups containing BG was observed, while there was a strong correlation between NDMA and the FG group cooked for 3 min. The use of BG caused an increase in NPIP, but affected NDMA and NDEA depending on the cooking time.