PBPK model-based gender-specific risk assessment of N-nitrosodimethylamine (NDMA) using human biomonitoring data.

Despite several screening levels for NDMA reported in water, soil, air, and drugs, the human risk assessment using biomonitoring concentrations has not been performed. In this study, gender-specific exposure guidance values were determined in humans, then biomonitoring measurements in healthy Korean subjects (32 men and 40 women) were compared to the exposure guidance values to evaluate the current exposure level to NDMA. For the human risk assessment of NDMA, the gender-specific physiologically based pharmacokinetic (PBPK) model was developed in humans using proper physiological parameters, partition coefficients, and biochemical parameters. Using the PBPK model, a Monte Carlo simulation was performed to describe the magnitudes of inter-individual variability and uncertainty on the single model predictions. The PBPK modeling and Monte Carlo simulation allowed the estimation of the relationship between external dose and blood concentration for the risk assessment. The procedure for the human risk assessment was summarized as follows: (1) estimating a steady-state blood concentration (Cavg) corresponding to the daily no observed adverse effect level (NOAEL) administration in rats; (2) applying uncertainty factors (UFs) for deriving the human Cavg; (3) determining the exposure guidance values as screening criteria; (4) interpreting the human biomonitoring measurements by forward and reverse dosimetry approaches. Using the biomonitoring concentrations, current daily exposures to NDMA were estimated to be 3.95 µg/day/kg for men and 10.60 µg/day/kg for women, respectively. The result of the study could be used as a basis for implementing further risk management and regulatory decision-making for NDMA.

Hepatoprotective effect of Holothuria leucospilota methanolic extract on dimethyl nitrosamine-induced hepatotoxicity in rats.

BACKGROUND: Complementary medicine is an interesting field for extracting bioactive compounds from various plant and animal sources. The hepatoprotective effect of the methanolic extract of a species of sea cucumber called Holothuria leucospilota in an animal model of liver cancer caused by dimethyl nitrosamine (DMN) was studied. METHODS: Wistar female rats were randomly divided into five groups (n = 12): control (intact), positive control (received 1% DMN [10 mg/kg/week, intraperitoneally] for 12 weeks), and three treatment groups (received 50, 100, and 200 mg/kg/day H. leucospilota extract orally for 12 weeks along with intraperitoneal administration of 1% DMN [10 mg/kg/week]). In all groups, ultrasound was performed on the liver every week to check its density. Blood sampling and liver isolation were performed on three occasions, at 4, 8, and 12 weeks, to check liver enzymes and the histopathological condition of the liver tissue (every week, four animals from each group were randomly selected). RESULTS: Liver density changes were evident from the eighth week onward in the positive control group. Histopathological results indicated pathologic changes in the positive control group after 4 weeks. The increase in liver enzymes in the positive control group was significantly different from that in the treatment and control groups. CONCLUSIONS: We demonstrated the hepatoprotective effect of H. leucospilota on DMN-induced liver damage in rats using biochemical and histological parameters and ultrasonography. More additional research (in silico or in vitro) is needed to find the exact mechanism and the main biological compound in H. leucospilota.

N-nitrosamine impurity risk assessment in pharmaceuticals: Utilizing In vivo mutation relative potency comparison to establish an acceptable intake for NTTP.

The finding of N-nitrosodiethylamine (NDEA) and N-nitrosodimethylamine (NDMA) in marketed drugs has led to implementation of risk assessment processes intended to limit exposures to the entire class of N-nitrosamines. A critical component of the risk assessment process is establishing exposure limits that are protective of human health. One approach to establishing exposure limits for novel N-nitrosamines is to conduct an in vivo transgenic rodent (TGR) mutation study. Existing regulatory guidance on N-nitrosamines provides decision making criteria based on interpreting in vivo TGR mutation studies as an overall positive or negative. However, point of departure metrics, such as benchmark dose (BMD), can be used to define potency and provide an opportunity to establish relevant exposure limits. This can be achieved through relative potency comparison of novel N-nitrosamines with model N-nitrosamines possessing robust in vivo mutagenicity and carcinogenicity data. The current work adds to the dataset of model N-nitrosamines by providing in vivo TGR mutation data for N-nitrosopiperidine (NPIP). In vivo TGR mutation data was also generated for a novel N-nitrosamine impurity identified in sitagliptin-containing products, 7-nitroso-3-(trifluoromethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo-[4,3-a]pyrazine (NTTP). Using the relative potency comparison approach, we have demonstrated the safety of NTTP exposures at or above levels of 1500 ng/day.

Commentary of the SKLM to the EFSA opinion on risk assessment of N-nitrosamines in food.

Dietary exposure to N-nitrosamines has recently been assessed by the European Food Safety Authority (EFSA) to result in margins of exposure that are conceived to indicate concern with respect to human health risk. However, evidence from more than half a century of international research shows that N-nitroso compounds (NOC) can also be formed endogenously. In this commentary of the Senate Commission on Food Safety (SKLM) of the German Research Foundation (DFG), the complex metabolic and physiological biokinetics network of nitrate, nitrite and reactive nitrogen species is discussed with emphasis on its influence on endogenous NOC formation. Pioneering approaches to monitor endogenous NOC have been based on steady-state levels of N-nitrosodimethylamine (NDMA) in human blood and on DNA adduct levels in blood cells. Further NOC have not been considered yet to a comparable extent, although their generation from endogenous or exogenous precursors is to be expected. The evidence available to date indicates that endogenous NDMA exposure could exceed dietary exposure by about 2-3 orders of magnitude. These findings require consolidation by refined toxicokinetics and DNA adduct monitoring data to achieve a credible and comprehensive human health risk assessment.

Evaluating the mutagenicity of N-nitrosodimethylamine in 2D and 3D HepaRG cell cultures using error-corrected next generation sequencing.

Human liver-derived metabolically competent HepaRG cells have been successfully employed in both two-dimensional (2D) and 3D spheroid formats for performing the comet assay and micronucleus (MN) assay. In the present study, we have investigated expanding the genotoxicity endpoints evaluated in HepaRG cells by detecting mutagenesis using two error-corrected next generation sequencing (ecNGS) technologies, Duplex Sequencing (DS) and High-Fidelity (HiFi) Sequencing. Both HepaRG 2D cells and 3D spheroids were exposed for 72 h to N-nitrosodimethylamine (NDMA), followed by an additional incubation for the fixation of induced mutations. NDMA-induced DNA damage, chromosomal damage, and mutagenesis were determined using the comet assay, MN assay, and ecNGS, respectively. The 72-h treatment with NDMA resulted in concentration-dependent increases in cytotoxicity, DNA damage, MN formation, and mutation frequency in both 2D and 3D cultures, with greater responses observed in the 3D spheroids compared to 2D cells. The mutational spectrum analysis showed that NDMA induced predominantly A:T → G:C transitions, along with a lower frequency of G:C → A:T transitions, and exhibited a different trinucleotide signature relative to the negative control. These results demonstrate that the HepaRG 2D cells and 3D spheroid models can be used for mutagenesis assessment using both DS and HiFi Sequencing, with the caveat that severe cytotoxic concentrations should be avoided when conducting DS. With further validation, the HepaRG 2D/3D system may become a powerful human-based metabolically competent platform for genotoxicity testing.

Trans-2-nonadecyl-4-(hydroxymethyl)-1,3-dioxolane (TNHD) purified from freshwater clams markedly alleviates dimethylnitrosamine-induced hepatic fibrosis.

Liver fibrosis occurs due to injury or inflammation, which results in the excessive production of collagen and the formation of fibrotic scar tissue that impairs liver function. Despite the limited treatment options available, freshwater clams may hold promise in the treatment of liver fibrosis. In this study, we demonstrated the effects of ethanol extract of freshwater clam (FCE), ethyl acetate extract of FCE (EA-FCE), and trans-2-nonadecyl-4-(hydroxymethyl)-1,3-dioxolane (TNHD) on liver fibrosis induced by dimethylnitrosamine (DMN). Administration of FCE and TNHD alleviated liver injury, including tissue damage, necrosis, inflammation scores, fibrosis scores, serum enzymes, and triglyceride levels. Furthermore, we analyzed the expression of fibrosis-related proteins, such as α-smooth muscle actin (α-SMA) and transforming growth factor (TGF-ß), as well as the hydroxyproline content, which decreased after treatment with FCE and TNHD. Animal experiments revealed that FCE and TNHD can reduce liver fibrosis by inhibiting cytokines that activate stellate cells and decreasing extracellular matrix (ECM) secretion. Cell experiments have shown that TNHD inhibits the MAPK/Smad signaling pathway and TGF-ß1 activation, resulting in a reduction in the expression of fibrosis-related proteins. Therefore, freshwater clam extracts, particularly TNHD, may have potential therapeutic and preventive effects for the amelioration of liver fibrosis.

Risk characterization of N-nitrosodimethylamine in pharmaceuticals.

Since 2018, N-nitrosodimethylamine (NDMA) has been a reported contaminant in numerous pharmaceutical products. To guide the pharmaceutical industry, FDA identified an acceptable intake (AI) of 96 ng/day NDMA. The approach assumed a linear extrapolation from the Carcinogenic Potency Database (CPDB) harmonic-mean TD50 identified in chronic studies in rats. Although NDMA has been thought to act as a mutagenic carcinogen in experimental animals, it has not been classified as a known human carcinogen by any regulatory agency. Humans are exposed to high daily exogenous and endogenous doses of NDMA. Due to the likelihood of a threshold dose for NDMA-related tumors in animals, we believe that there is ample scientific basis to utilize the threshold-based benchmark dose or point-of-departure (POD) approach when estimating a Permissible Daily Exposure limit (PDE) for NDMA. We estimated that 29,000 ng/kg/day was an appropriate POD for calculating a PDE. Assuming an average bodyweight of 50 kg, we expect that human exposures to NDMA at doses below 5800 ng/day in pharmaceuticals would not result in an increased risk of liver cancer, and that there is little, if any, risk for any other type of cancer, when accounting for the mode-of-action in humans.

Effect of Niosomal Encapsulation of Quercetin and Silymarin and their Combination on Dimethylnitrosoamine-induced and Phenobarbital promoted Hepatocellular Carcinoma in Rat Model.

BACKGROUND: Hepatocellular carcinoma is a particularly dangerous and severe kind of liver cancer. Many anticancer drugs fail to complete the treatment of hepatocellular carcinoma without any side effects. There should be appropriate and without side effective treatments for hepatocellular carcinoma. OBJECTIVE: The objective of the current study was to evaluate how quercetin and silymarin in a niosomal formulation affected hepatocyte carcinoma caused by diethylnitrosamine. METHODS: Five groups were created from the thirty male rats. Normal control (untreated group), tumor group (administered dimethylnitrosoamine 200 mg/kg), treatment group I (administered 50 mg/kg of niosomal encapsulated quercetin), treatment group II (administered 50 mg/kg of niosomal encapsulated silymarin), and treatment group III (administered 50 mg/kg of niosomal encapsulated quercetin + silymarin). Then, biochemical estimation, serum analysis, and histopathological examination were carried out. RESULTS: Treatment group III, treated with niosomal encapsulation of a combination of quercetin + silymarin 50 mg/kg, demonstrated the significant restoration of alpha-fetoprotein and carcinoembryonic antigen and also antioxidants like superoxide dismutase and nitric oxide. The histopathological examination showed improved liver architecture in this group compared to other treatment groups. CONCLUSION: Our findings revealed that a potent anticancer effect was observed in treatment group III as niosomal formulation increased the bioavailability of the drug within the body. In order to completely understand the underlying processes and evaluate the therapeutic effectiveness of these chemicals in the therapy of hepatocellular carcinoma, further investigation and clinical trials are required.

N-Nitrosodimethylamine investigations in Muta™Mouse define point-of-departure values and demonstrate less-than-additive somatic mutant frequency accumulations.

The N-nitrosamine, N-nitrosodimethylamine (NDMA), is an environmental mutagen and rodent carcinogen. Small levels of NDMA have been identified as an impurity in some commonly used drugs, resulting in several product recalls. In this study, NDMA was evaluated in an OECD TG-488 compliant Muta™Mouse gene mutation assay (28-day oral dosing across seven daily doses of 0.02-4 mg/kg/day) using an integrated design that assessed mutation at the transgenic lacZ locus in various tissues and at the endogenous Pig-a gene-locus, along with micronucleus frequencies in peripheral blood. Liver pathology was determined together with NDMA exposure in blood and liver. The additivity of mutation induction was assessed by including two acute single-dose treatment groups (i.e. 5 and 10 mg/kg dose on Day 1), which represented the same total dose as two of the repeat dose treatment groups. NDMA did not induce statistically significant increases in mean lacZ mutant frequency (MF) in bone marrow, spleen, bladder, or stomach, nor in peripheral blood (Pig-a mutation or micronucleus induction) when tested up to 4 mg/kg/day. There were dose-dependent increases in mean lacZ MF in the liver, lung, and kidney following 28-day repeat dosing or in the liver and kidney after a single dose (10 mg/kg). No observed genotoxic effect levels (NOGEL) were determined for the positive repeat dose-response relationships. Mutagenicity did not exhibit simple additivity in the liver since there was a reduction in MF following NDMA repeat dosing compared with acute dosing for the same total dose. Benchmark dose modelling was used to estimate point of departure doses for NDMA mutagenicity in Muta™Mouse and rank order target organ tissue sensitivity (liver > kidney or lung). The BMD50 value for liver was 0.32 mg/kg/day following repeat dosing (confidence interval 0.21-0.46 mg/kg/day). In addition, liver toxicity was observed at doses of ≥ 1.1 mg/kg/day NDMA and correlated with systemic and target organ exposure. The integration of these results and their implications for risk assessment are discussed.

Physiologically-based pharmacokinetic model for evaluating gender-specific exposures of N-nitrosodimethylamine (NDMA).

N-nitrosodimethylamine (NDMA) is classified as a human carcinogen and could be produced by both natural and industrial processes. Although its toxicity and histopathology have been well-studied in animal species, there is insufficient data on the blood and tissue exposures that can be correlated with the toxicity of NDMA. The purpose of this study was to evaluate gender-specific pharmacokinetics/toxicokinetics (PKs/TKs), tissue distribution, and excretion after the oral administration of three different doses of NDMA in rats using a physiologically-based pharmacokinetic (PBPK) model. The major target tissues for developing the PBPK model and evaluating dose metrics of NDMA included blood, gastrointestinal (GI) tract, liver, kidney, lung, heart, and brain. The predictive performance of the model was validated using sensitivity analysis, (average) fold error, and visual inspection of observations versus predictions. Then, a Monte Carlo simulation was performed to describe the magnitudes of inter-individual variability and uncertainty of the single model predictions. The developed PBPK model was applied for the exposure simulation of daily oral NDMA to estimate blood concentration ranges affecting health effects following acute-duration (≤ 14 days), intermediate-duration (15-364 days), and chronic-duration (≥ 365 days) intakes. The results of the study could be used as a scientific basis for interpreting the correlation between in vivo exposures and toxicological effects of NDMA.

Genotoxicity assessment of eight nitrosamines using 2D and 3D HepaRG cell models.

N-nitrosamine impurities have been increasingly detected in human drugs. This is a safety concern as many nitrosamines are mutagenic in bacteria and carcinogenic in rodent models. Typically, the mutagenic and carcinogenic activity of nitrosamines requires metabolic activation by cytochromes P450 enzymes (CYPs), which in many in vitro models are supplied exogenously using rodent liver homogenates. There are only limited data on the genotoxicity of nitrosamines in human cell systems. In this study, we used metabolically competent human HepaRG cells, whose metabolic capability is comparable to that of primary human hepatocytes, to evaluate the genotoxicity of eight nitrosamines [N-cyclopentyl-4-nitrosopiperazine (CPNP), N-nitrosodibutylamine (NDBA), N-nitrosodiethylamine (NDEA), N-nitrosodimethylamine (NDMA), N-nitrosodiisopropylamine (NDIPA), N-nitrosoethylisopropylamine (NEIPA), N-nitroso-N-methyl-4-aminobutyric acid (NMBA), and N-nitrosomethylphenylamine (NMPA)]. Under the conditions we used to culture HepaRG cells, three-dimensional (3D) spheroids possessed higher levels of CYP activity compared to 2D monolayer cells; thus the genotoxicity of the eight nitrosamines was investigated using 3D HepaRG spheroids in addition to more conventional 2D cultures. Genotoxicity was assessed as DNA damage using the high-throughput CometChip assay and as aneugenicity/clastogenicity in the flow-cytometry-based micronucleus (MN) assay. Following a 24-h treatment, all the nitrosamines induced DNA damage in 3D spheroids, while only three nitrosamines, NDBA, NDEA, and NDMA, produced positive responses in 2D HepaRG cells. In addition, these three nitrosamines also caused significant increases in MN frequency in both 2D and 3D HepaRG models, while NMBA and NMPA were positive only in the 3D HepaRG MN assay. Overall, our results indicate that HepaRG spheroids may provide a sensitive, human-based cell system for evaluating the genotoxicity of nitrosamines.

Protocatechuic acid modulates hepatic oxidative stress and inflammation linked to DMN exposure in rat.

Dimethyl nitrosamine (DMN), a potent hepatotoxin, exerts carcinogenic effects and induces hepatic necrosis in experimental animals via CYP2E1 metabolic activation, and generation of reactive oxygen species (ROS). Protocatechuic acid (PCA), a plant-based simple phenolic compound and potent antioxidant, has been shown to affect the development of neoplasia in the rat liver and inhibit the initiation or progression phases of most cancers. In this study, the modulatory effects of PCA on DMN-induced hepatotoxicity, oxidative stress, inflammation, and selected phase I xenobiotic metabolizing enzymes were investigated in male Wistar rats. This study assessed biomarkers of hepatic injury (alanine transaminase, aspartate aminotransferase, alkaline phosphatase, and gamma- glutamyl transferase); oxidative stress (hydrogen peroxide concentration, lipid peroxidation, and reduced glutathione levels); measured activities of antioxidant enzymes (catalase, sodium dismutase, glutathione peroxidase, glutathione S-transferase); and inflammation (Tumor necrosis factor (TNF)-α, interleukin-1-Beta (IL-1ß) and iNOS). The results of our investigation demonstrated that pretreatment with PCA at 50 and 100 mg/kg body weight p.o. reduced DMN (20 mg/kg bw) i.p. mediated hepatic injury, oxidative stress, and inflammation in a dose-dependent manner. In addition, the activities of phase I metabolizing enzymes were significantly induced except for aminopyrine-N-demethylase in the DMN-treated rats when compared with the DMN alone control group. This induction was also reversed by pre-treatment with PCA. The result of this study suggests that PCA is hepatoprotective against DMN-induced hepatic damage by its ability to suppress oxidative stress, inflammation, and modulate the activities of the selected phase I drug metabolizing enzymes. Thus, PCA may prove useful in combating DMN-induced hepatic damage.

Pharmacoepidemiological Research on N-Nitrosodimethylamine-Contaminated Ranitidine Use and Long-Term Cancer Risk: A Population-Based Longitudinal Cohort Study.

N-Nitrosodimethylamine (NDMA), a carcinogenic chemical, has recently been identified in ranitidine. We conducted a population-based study to explore ranitidine use and cancer emergence over time. Using the Taiwan National Health Insurance Research Database, a population-based cohort study was conducted. A total of 55,110 eligible patients who received ranitidine between January 2000 and December 2018 were enrolled in the treated cohort. We conducted a 1:1 propensity-score-matching procedure to match the ranitidine-treated group with the ranitidine-untreated group and famotidine controls for a longitudinal study. The association of ranitidine exposure with cancer outcomes was assessed. A multivariable Cox regression analysis that compared cancer risk with the untreated groups revealed that ranitidine increased the risk of liver (hazard ratio (HR): 1.22, 95% confidence interval (CI): 1.09-1.36, p < 0.001), lung (HR: 1.17, CI: 1.05-1.31, p = 0.005), gastric (HR: 1.26, CI: 1.05-1.52, p = 0.012), and pancreatic cancers (HR 1.35, CI: 1.03-1.77, p = 0.030). Our real-world observational study strongly supports the pathogenic role of NDMA contamination, given that long-term ranitidine use is associated with a higher likelihood of liver cancer development in ranitidine users compared with the control groups of non-ranitidine users treated with famotidine or proton-pump inhibitors.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) shows therapeutic effect on dimethylnitrosamine (DMN)-induced liver fibrosis in rats.

This study was undertaken to investigate the inhibitory effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) on dimethylnitrosamine (DMN)-induced liver fibrosis in rats. Liver fibrosis was induced in Sprague-Dawley rats by injecting DMN intraperitoneally (at 10 mg/kg of body weight) daily for three consecutive days per week for 4 weeks. To investigate the effect of GM-CSF on disease onset, GM-CSF (50 µg/kg of body weight) was co-treated with DMN for 2 consecutive days per week for 4 weeks (4-week groups). To observe the effect of GM-CSF on the progression of liver fibrosis, GM-CSF was post-treated alone at 5-8 weeks after the 4 weeks of DMN injection (8-week groups). We found that DMN administration for 4 weeks produced molecular and pathological manifestations of liver fibrosis, that is, it increased the expressions of collagen type I, alpha-smooth muscle actin (α-SMA), and transforming growth factor-ß1 (TGF-ß1), and decreased peroxisome proliferator-activated receptor gamma (PPAR-γ) expression. In addition, elevated serum levels of aspartate aminotransferase (AST), total bilirubin level (TBIL), and decreased albumin level (ALB) were observed. In both the 4-week and 8-week groups, GM-CSF clearly improved the pathological liver conditions in the gross and histological observations, and significantly recovered DMN-induced increases in AST and TBIL and decreases in ALB serum levels to normal. GM-CSF also significantly decreased DMN-induced increases in collagen type I, α-SMA, and TGF-ß1 and increased DMN-induced decreases in PPAR-γ expression. In the DMN groups, survival decreased continuously for 8 weeks after DMN treatment for the first 4 weeks. GM-CSF showed a survival benefit when co-treated for the first 4 weeks but a marginal effect when post-treated for 5-8 weeks. In conclusion, co-treatment of GM-CSF showed therapeutic effects on DMN-induced liver fibrosis and survival rates in rats, while post-treatment efficiently blocked liver fibrosis.

Analysis of genotoxic N-nitrosamines in active pharmaceutical ingredients and market authorized products in low abundance by means of liquid chromatography - tandem mass spectrometry.

In 2018, high levels of the IARC class IIA carcinogen N-nitrosodimethylamine (NDMA) were analytically verified in the active pharmaceutical ingredient (API) valsartan, resulting in extensive regulatory action on angiotensin-II-receptor antagonists and recall of finished drug products by the pharmaceutical industry to ensure patient safety. The root cause of contamination was the unintended reaction of common reagents utilized during drug synthesis. This lead to serious effects on drug quality and immediate regulatory action. Thus, routine analysis of drug product contents are inevitable and necessitate thoroughly performed work up procedures of the product as well as adequate validated analytical methods. The nature of N-nitrosamines (NA), ranging from small, semi-volatile compounds up to highly polar molecules, effort sophisticated requirements in terms of instrumental analysis. Up today, gas as well as liquid chromatographic devices coupled to mass spectrometers are the most widespread systems for analysis. Gas chromatographic - mass spectrometric (GC-MS) systems, obviously superior towards liquid chromatography - mass spectrometry (LC-MS) for detecting small volatile compounds like NDMA, reach their limits for broadly designed studies including polar or acidic NA. In this study, a complementary and highly sensitive approach by means of liquid chromatography - tandem mass spectrometry (LC-MS/MS) is presented, including detection of 13 NA deduced from major classes of secondary amines. Thereby, the fully validated approach was performed in accordance to ICH and European Medicines Agency (EMA) guidelines. Quantitative proof-of-concept measurements with various APIs and market authorized tablets as representative drug formulations conclude applicability for further presumably contaminated substances. The approach employs organic or inorganic extraction steps with solid phase extraction (SPE). The limit of detection for the most prominent NA, NDMA and N-diethylnitrosamine (NDEA), were both 0.025 parts-per-billion (ppb) per matrix, respectively.

Estimated Cancer Risks Associated with Nitrosamine Contamination in Commonly Used Medications.

Many nitrosamines are potent carcinogens, with more than 30 listed under California's Proposition 65. Recently, nitrosamine contamination of commonly used drugs for treatment of hypertension, heartburn, and type 2 diabetes has prompted numerous Food and Drug Administration (FDA) recalls in the US. These contaminants include the carcinogens NDMA (N-nitrosodimethylamine) and NDEA (N-nitrosodiethylamine) and the animal tumorigen NMBA (N-nitroso-N-methyl-4-aminobutyric acid). NMBA and NDEA are metabolically and/or structurally related to NDMA, an N-nitrosomethyl-n-alkylamine (NMA), and 12 other carcinogenic NMAs. These nitrosamines exhibit common genotoxic and tumorigenic activities, with shared target tumor sites amongst chemicals and within a given laboratory animal species. We use the drug valsartan as a case study to estimate the additional cancer risks associated with NDMA and NDEA contamination, based on nitrosamine levels reported by the US FDA, cancer potencies developed by California's Proposition 65 program and the US Environmental Protection Agency (EPA), and specific exposure scenarios. These estimates suggest that nitrosamine contamination in drugs that are used long-term can increase cancer risks and pose a serious concern to public health.

Betulinic acid abates N-nitrosodimethylamine-induced changes in lipid metabolism, oxidative stress, and inflammation in the liver and kidney of Wistar rats.

N-nitrosamines have been linked with cancer in humans due to their presence in drinking water and diets. This study evaluated the role of betulinic acid (BA) in abating oxidative stress, inflammation, and hyperlipidemia in rats treated with N-nitrosodimethylamine (NDMA). Twenty-four male rats were assigned into four equal groups. Group I served as the control, Group II received BA (25 mg/kg), Group III received NDMA (5 mg/kg) and, Group IV received BA (25 mg/kg) and NDMA (5 mg/kg). Results showed that the administration of NDMA significantly (p < 0.05) elevated malondialdehyde in the liver and kidney relative to controls. Activities of superoxide dismutase, catalase, glutathione peroxidase, and glutathione-S-transferase, and the level of glutathione were significantly (p < 0.05) decreased by NDMA, while treatment with BA elevated the activities of these enzymes in the liver and kidney. The BA lowered serum interleukin-6 and tumor necrosis factor-alpha levels against the NDMA effect. Furthermore, NDMA increased hepatic and renal triglyceride while phospholipids levels were decreased. NDMA significantly modulated the activities of drug-metabolizing enzymes (aniline hydroxylase, aminopyrine-N-demethylase, and uridyldiphosphoglucuronyltransferase), while BA was able to restore these enzymes to values close to controls. Histology revealed the presence of infiltration and fibroplasia in the liver, while cortical degeneration was noticed in the kidney in NDMA-administered rats. These lesions were reduced in the NDMA rats treated with BA. The findings suggest that BA improves NDMA-induced damage in the liver and kidney of rats through reactions that can be linked with antioxidant, anti-inflammatory, and lipid-lowering pathways.

CagA+ Helicobacter pylori infection and N-nitrosodimethylamine administration induce cholangiocarcinoma development in hamsters.

BACKGROUND: Helicobacter pylori (HP) has been detected in the hepatobiliary tract of cholangiocarcinoma (CCA) patients in regions both endemic and non-endemic for Opisthorchis viverrini (OV) infection. However, whether H. pylori infection promotes CCA development remains unknown. We investigated CCA development in hamsters induced by a combination of infection with H. pylori and administration of N-nitrosodimethylamine (NDMA) and compared findings with those in an OV plus NDMA group. MATERIALS AND METHODS: Eighty-five hamsters were divided into four groups: (1) normal, (2) administered NDMA, (3) infected with cagA+ H. pylori and administered NDMA (HN group), and (4) infected with OV and administered NDMA (ON group). Animals were euthanized at 3 and 6 months post-infection. Histopathological changes of liver and the expression of markers associated with carcinogenesis were studied. RESULTS: At 3 months post-infection (p.i.), cholangitis and lymphoid follicles without tumor appearance were noted in the HN group, whereas extensive fibrosis was seen in members of the ON group, 10% of which had developed tumors. At 6 months p.i., 10% of hamsters administered NDMA alone had developed CCA, whereas in the HN and ON groups, 20% and 60% of hamsters, respectively, had developed CCA. Cytokeratin-19 (CK19) expression was observed in the CCA tissues of both the HN and the ON groups, confirming the bile duct origin of the CCA cells. CCA development in the HN group might be inflammation-mediated, as suggested by overexpression of HMGB1, PCNA, IL-8, and 8-OxodG in CCA tissues. CONCLUSION: cagA+ H. pylori infection and carcinogen intake can induce CCA development with slow progression.

MiR-34c promotes hepatic stellate cell activation and Liver Fibrogenesis by suppressing ACSL1 expression.

Normally, there are multiple microRNAs involved in the pathogenesis of liver fibrosis. In our work, we aimed at identifying the role of miR-34c in the hepatic stellate cell (HSC) activation and liver fibrosis and its potential mechanism. Our results have shown that during natural activation of HSC, the level of miR-34c was increased significantly whereas acyl-CoA synthetase long-chain family member-1(ACSL1), which is a key enzyme can affect fatty acid(FA) synthesis, was decreased. A double fluorescence reporter assay further confirmed that ACSL1 is a direct target gene of miR-34c. Moreover, the inhibition of miR-34C can attenuate the synthesis of collagen in HSC-T6. In our rescue assay, ACSL1 expression was 1.49-fold higher compared to normal control cells which were transfected with the miR-34c inhibitor in a stable low expression ACSL1 cell line. While at the same time, α-SMA and Col1α expression decreased by 18.22% and 2.58%, respectively. Moreover, we performed an in vivo model using dimethylnitrosamine (DMN) in conjunction with the miR-34c agomir, combined with the treatment of DMN and the miR-34c agomir can increase liver fibrosis. Meanwhile, the degree of hepatic fibrosis was increased and lipid droplets reduced dramatically in rats and HSC-T6 cell treated with miR-34c mimics alone compared to untreated groups. Our results indicate that miR-34c plays an essential role in liver fibrosis by targeting ACSL1 closely associated with lipid droplets, and it might be used as a potential therapeutic target.

Hepatoprotective effect of genistein against dimethylnitrosamine-induced liver fibrosis in rats by regulating macrophage functional properties and inhibiting the JAK2/STAT3/SOCS3 signaling pathway.

BACKGROUND: Liver fibrosis is a dysregulated wound-healing process in response to diverse liver injuries, and an effective drug therapy is not yet available. Genistein, which is one of the most active natural flavonoids mainly derived from soybean products (e.g., Cordyceps sinensis mycelium), exhibits various biological effects, including hepatoprotective and anti-inflammatory properties. However, the anti-hepatic fibrosis mechanisms of genistein are poorly understood. The aim of our research is to explore the effect and the possible mechanism of genistein against liver fibrosis. MATERIALS AND METHODS: Cell counting kit-8, EdU, and flow cytometry assays were applied to evaluate the effects of genistein on cell viability, proliferation, and cell cycle arrest in human hepatic stellate cell (HSC) line LX2 cells. HSC activation was induced by transforming growth factor-ß1 in LX2 cells and liver fibrosis model was established by the intraperitoneal injection of dimethylnitrosamine (DMN) in rats to assess the anti-fibrosis effects of genistein in vivo and in vitro models. HSC activation was assessed by qRT-PCR, Western blot, immunohistochemistry, and immunofluorescent assay. Liver injury and collagen deposition were evaluated by histopathological assay, serum biochemistry, and hepatic hydroxyproline content assays. The mRNA expressions of matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), and inflammation related-factors were assessed by qRT-PCR assay. Furthermore, the functional properties of macrophage in the liver were assessed by immunohistochemistry assay. The expression levels of the JAK2/STAT3/SOCS3 signaling pathway related-protein were assessed by Western blot analysis. RESULTS: Genistein significantly inhibited cell viability and proliferation and induced cell cycle arrest at G0/G1 phase in LX2 cells, respectively. Furthermore, oral administration of genistein significantly ameliorated liver injury and the collagen deposition in rats with DMN-induced fibrosis model. Genistein suppressed the expression levels of HSC activation marker α-smooth muscle actin and collagen type I alpha 1 in vivo and in vitro. Genistein significantly decreased the mRNA expression levels of extracellular matrix degradation genes MMP2/9 and TIMP1 in rats. Genistein alleviated the mRNA expression levels of IL-1ß, IL-6, TNF-α, and MCP-1 and regulated the protein expressions of CD68, CD163, and CD206 in the liver. Moreover, genistein attenuated the expressions of p-JAK2/JAK2, p-STAT3/STAT3, and SOCS3 protein both in vivo and in vitro. CONCLUSION: Taken together, our results showed that genistein could be improved liver fibrosis both in vivo and in vitro, probably through regulating the functional properties of macrophage and inhibiting the JAK2/STAT3/SOCS3 signaling pathway.