N-nitrosodimethylamine exposure to zebrafish embryos/larvae causes cardiac and spinal developmental toxicity.

N-nitrosodimethylamine (NDMA), one of the new nitrogen-containing disinfection by-products, is potentially cytotoxic, genotoxic, and carcinogenic. Its potential toxicological effects have attracted a wide range of attention, but the mechanism is still not sufficiently understood. To better understand the toxicological mechanisms of NDMA, zebrafish embryos were exposed to NDMA from 3 h post-fertilization (hpf) to 120hpf. Mortality and malformation were significantly increased, and hatching rate, heart rate, and swimming behavior were decreased in the exposure groups. The result indicated that NDMA exposure causes cardiac and spinal developmental toxicity. mRNA levels of genes involved in the apoptotic pathway, including p53, bax, and bcl-2 were significantly affected by NDMA exposure. Moreover, the genes associated with spinal and cardiac development (myh6, myh7, nkx2.5, eph, bmp2b, bmp4, bmp9, run2a, and run2b) were significantly downregulated after treatment with NDMA. Wnt and TGF-ß signaling pathways, crucial for the development of diverse tissues and organs in the embryo and the establishment of the larval spine, were also significantly disturbed by NDMA treatment. In summary, the disinfection by-product, NDMA, exhibits spinal and cardiac developmental toxicity in zebrafish embryos, providing helpful information for comprehensive analyses and a better understanding the mechanism of its toxicity.

NDMA Formation Due to Active Ingredient Degradation and Nitrite Traces in Drug Product.

N-nitrosamines are genotoxic compounds which can be found as impurities in drug substances and drug products used in the pharmaceutical industry. To date, several possible nitrosamine sources in drug products have been reported and this study aims to illuminate another one. A case of afatinib drug product was investigated, in which up to 50 ppb N-nitrosodimethylamine (NDMA) traces were detected. Afatinib was found to degrade to the secondary amine dimethylamine (DMA), forming NDMA with traces of nitrite in crospovidone. Two series of film-coated tablets were prepared with crospovidone from two different manufacturers, containing different levels of nitrites. Tablets were subjected to an accelerated stability study (40 °C/75% relative humidity) or stored at room temperature and levels of NDMA, DMA and nitrite in tablets were monitored. NDMA and nitrite were found on ppb levels, whereas DMA was detected on ppm levels. NDMA formation in the drug product was found to be time, temperature and nitrite dependent and it was emphasized that DMA and nitrite should be reduced. The accelerated stability study proved to be a useful tool for predicting nitrosamine formation in the drug product.

In Vitro Analysis of N-Nitrosodimethylamine (NDMA) Formation From Ranitidine Under Simulated Gastrointestinal Conditions.

Importance: A publication reported that N-nitrosodimethylamine (NDMA), a probable human carcinogen, was formed when ranitidine and nitrite were added to simulated gastric fluid. However, the nitrite concentrations used were greater than the range detected in acidic gastric fluid in prior clinical studies. Objective: To characterize NDMA formation following the addition of ranitidine to simulated gastric fluid using combinations of fluid volume, pH levels, and nitrite concentrations, including physiologic levels. Design, Setting, and Participants: One 150-mg ranitidine tablet was added to 50 or 250 mL of simulated gastric fluid with a range of nitrite concentrations from the upper range of physiologic (100 µmol/L) to higher concentrations (10 000 µmol/L) with a range of pH levels. NDMA amounts were assessed with a liquid chromatography-mass spectrometry method. Main Outcomes and Measures: NDMA detected in simulated gastric fluid 2 hours after adding ranitidine. Results: At a supraphysiologic nitrite concentration (ie, 10 000 µmol/L), the mean (SD) amount of NDMA detected in 50 mL simulated gastric fluid 2 hours after adding ranitidine increased from 222 (12) ng at pH 5 to 11 822 (434) ng at pH 1.2. Subsequent experiments with 50 mL of simulated gastric fluid at pH 1.2 with no added nitrite detected a mean (SD) of 22 (2) ng of NDMA, which is the background amount present in the ranitidine tablets. Similarly, at the upper range of physiologic nitrite (ie, 100 µmol/L) or at nitrite concentrations as much as 50-fold greater (1000 or 5000 µmol/L) only background mean (SD) amounts of NDMA were observed (21 [3] ng, 24 [2] ng, or 24 [3] ng, respectively). With 250 mL of simulated gastric fluid, no NDMA was detected at the upper physiologic range (100 µmol/L) or 10-fold physiologic (1000 µmol/L) nitrite concentrations, while NDMA was detected (mean [SD] level, 7353 [183] ng) at a 50-fold physiologic nitrite concentration (5000 µmol/L). Conclusions and Relevance: In this in vitro study of ranitidine tablets added to simulated gastric fluid with different nitrite concentrations, ranitidine conversion to NDMA was not detected until nitrite was 5000 µmol/L, which is 50-fold greater than the upper range of physiologic gastric nitrite concentrations at acidic pH. These findings suggest that ranitidine is not converted to NDMA in gastric fluid at physiologic conditions.

Comparative disposition of dimethylaminoethanol and choline in rats and mice following oral or intravenous administration.

Dimethylaminoethanol (DMAE) and its salts have been used to treat numerous disorders in humans and hence safety of its use is a concern. DMAE is a close structural analog of choline, an essential nutrient. Exposure to DMAE may affect choline uptake and synthesis. The current investigation characterizes: 1) the absorption, distribution, metabolism, and excretion (ADME) of DMAE in Wistar Han rats and B6C3F1 mice following a single gavage or intravenous (IV) administration of 10, 100 or 500 mg/kg [14C]DMAE, and 2) the ADME of [14C]choline (160 mg/kg) and the effect on its disposition following pre-treatment with DMAE (100 or 500 mg/kg). In both rats and mice, following gavage administration, DMAE was excreted in urine (16-69%) and as exhaled CO2 (3-22%). The tissue retention was moderate (21-44%); however, the brain concentrations were low and there was no accumulation. Serum choline levels were not elevated following administration of DMAE. The DMAE metabolites in urine were DMAE N-oxide and N,N-dimethylglycine; the carcinogen, N-N-dimethylnitrosamine, was not detected. The pattern of disposition of [14C]choline following gavage administration was similar to that of [14C]DMAE. Prior treatment with DMAE had minimal effects on choline disposition. The pattern of disposition of [14C]DMAE and [14C]choline following IV administration was similar to gavage administration. There were minimal dose-, sex- or species-related effects following gavage or IV administration of [14C]DMAE or [14C]choline. Data from the current study did not support previous reports that: 1) DMAE alters choline uptake and distribution, or 2) that DMAE is converted into choline in vivo.

Molecular mechanisms in the pathogenesis of N-nitrosodimethylamine induced hepatic fibrosis.

Hepatic fibrosis is marked by excessive synthesis and deposition of connective tissue proteins, especially interstitial collagens in the extracellular matrix of the liver. It is a result of an abnormal wound healing in response to chronic liver injury from various causes such as ethanol, viruses, toxins, drugs, or cholestasis. The chronic stimuli involved in the initiation of fibrosis leads to oxidative stress and generation of reactive oxygen species that serve as mediators of molecular events involved in the pathogenesis of hepatic fibrosis. These processes lead to cellular injury and initiate inflammatory responses releasing a variety of cytokines and growth factors that trigger activation and transformation of resting hepatic stellate cells into myofibroblast like cells, which in turn start excessive synthesis of connective tissue proteins, especially collagens. Uncontrolled and extensive fibrosis results in distortion of lobular architecture of the liver leading to nodular formation and cirrhosis. The perpetual injury and regeneration process could also results in genomic aberrations and mutations that lead to the development of hepatocellular carcinoma. This review covers most aspects of the molecular mechanisms involved in the pathogenesis of hepatic fibrosis with special emphasize on N-Nitrosodimethylamine (NDMA; Dimethylnitorsmaine, DMN) as the inducing agent.

Fungal biodegradation of the N-nitrosodimethylamine precursors venlafaxine and O-desmethylvenlafaxine in water.

Antidepressant drugs such as Venlafaxine (VFX) and O-desmethylvenlafaxine (ODMVFX) are emerging contaminants that are commonly detected in aquatic environments, since conventional wastewater treatment plants are unable to completely remove them. They can be precursors of hazardous by-products, such as the carcinogenic N-nitrosodimethylamine (NDMA), generated upon water chlorination, as they contain the dimethylamino moiety, necessary for the formation of NDMA. In this study, the capability of three white rot fungi (Trametes versicolor, Ganoderma lucidum and Pleurotus ostreatus) to remove both antidepressants from water and to decrease NDMA formation potential was investigated. Furthermore, transformation by-products (TPs) generated along the treatment process were elucidated and also correlated with their NDMA formation potential. Very promising results were obtained for T. versicolor and G. lucidum, both being able to remove up to 100% of ODMVFX. In the case of VFX, which is very recalcitrant to conventional wastewater treatment, a 70% of removal was achieved by T. versicolor, along with a reduction in NDMA formation potential, thus decreasing the associated problems for human health and the environment. However, the NDMA formation potential remained practically constant during treatment with G. lucidum despite of the equally high VFX removal (70%). This difference was attributed to the generation of different TPs during both fungal treatments. For example, G. lucidum generated more ODMVFX, which actually has a higher NDMA formation potential than the parent compound itself.

Biological treatment of N-nitrosodimethylamine (NDMA) and N-nitrodimethylamine (NTDMA) in a field-scale fluidized bed bioreactor.

The ex situ treatment of N-nitrosodimethylamine (NDMA) and N-nitrodimethylamine (NTDMA) in groundwater was evaluated in a field-scale fluidized bed bioreactor (FBR). Both of these compounds, which originally entered groundwater at the test site from the use of liquid rocket propellant, are suspected human carcinogens. The objective of this research was to examine the application of a novel field-scale propane-fed fluidized bed bioreactor as an alternative to ultraviolet irradiation (UV) for treating NDMA and NTDMA to low part-per-trillion (ng/L) concentrations. Previous laboratory studies have shown that the bacterium Rhodococcus ruber ENV425 can biodegrade NDMA and NTDMA during growth on propane as a primary substrate and that the strain can effectively reduce NDMA concentrations in propane-fed bench-scale bioreactors of different design. R. ruber ENV425 was used as a seed culture for the FBR, which operated at a fluidization flow of ∼19 L-per-min (LPM) and received propane, oxygen, and inorganic nutrients in the feed. The reactor effectively treated ∼1 µg/L of influent NDMA to effluent concentrations of less than 10 ng/L at a hydraulic residence time (HRT) of only 10 min. At a 20 min HRT, the FBR reduced NDMA to <4.2 ng/L in the effluent, which was the discharge limit at the test site where the study was conducted. Similarly, NTDMA was consistently treated in the FBR from ∼0.5 µg/L to <10 ng/L at an HRT of 10 min or longer. Based on these removal rates, the average NDMA and NTDMA elimination capacities achieved were 2.1 mg NDMA treated/m3 of expanded bed/hr of operation and 1.1 mg NTDMA treated/m3 of expanded bed/hr of operation, respectively. The FBR system was highly resilient to upsets including power outages. Treatment of NDMA, but not NTDMA, was marginally affected when trace co-contaminants including trichloroethene (TCE) and trichlorofluoromethane (Freon 11) were initially added to feed groundwater, but performance recovered over a few weeks in the continued presence of these compounds. Strain ENV425 appeared to be replaced by native propanotrophs over time based on qPCR analysis, but contaminant treatment was not diminished. The results suggest that a FBR can be a viable alternative to UV treatment for removing NDMA from groundwater.

Experimental and Theoretical Investigation of Effects of Ethanol and Acetic Acid on Carcinogenic NDMA Formation in Simulated Gastric Fluid.

N-nitrosodimethylamine (NDMA), as a representative of endogenously formed N-nitroso compounds (NOCs), has become the focus of considerable research interest due to its unusually high carcinogenicity. In this study, effects of ethanol and acetic acid on the formation of NDMA from dimethylamine (DMA) and nitrite in simulated gastric fluid (SGF) were investigated. Experimental results showed that ethanol in the concentrations of 1-8% (v/v) and acetic acid in the concentrations of 0.01-8% (v/v) exhibit inhibitory and promotion effects on the formation of NDMA, respectively. Moreover, they are both in a dose-dependent manner with the largest inhibition/promotion rate reaching ∼70%. Further experimental investigations indicate that ethanol and acetic acid are both able to scavenge nitrite in SGF. It implies that there are interactions of ethanol and acetic acid with nitrite or nitrite-related nitrosating agents rather than DMA. Theoretical calculations confirm the above experimental results and demonstrate that ethanol and acetic acid can both react with nitrite-related nitrosating agents to produce ethyl nitrite (EtONO) and acetyl nitrite (AcONO), respectively. Furthermore, the reactivities of ethyl nitrite, acetyl nitrite, and dinitrogen trioxide reacting with DMA were found in the order of AcONO > N2O3 ≫ EtONO. This is probably the main reason why there are completely different effects of ethanol and acetic acid on NDMA formation. On the basis of the above results, two requirements for a potential inhibitor of NOCs formation in SGF were provided. The results obtained in this study will be helpful in better understanding the inhibition/promotion mechanisms of compounds on NDMA formation in SGF and searching for protective substances to prevent carcinogenic NOCs formation.

Cytotoxic and genotoxic profiles of benzo[a]pyrene and N-nitrosodimethylamine demonstrated using DNA repair deficient DT40 cells with metabolic activation.

Benzo[a]pyrene and N-nitrosodimethylamine are major genotoxic compounds present in cigarette smoke, food and oil. To examine the type(s) of DNA damage induced by these compounds, we used a panel of DNA-repair-pathway-deficient mutants generated from chicken DT40 cells and achieved metabolic activation of the test compounds by including rat liver S9 mix. Consistent with expections, benzo[a]pyrene and N-nitrosodimethylamine require metabolicactivation to become genotoxic. The REV3(-/-) mutant cell line exhibited the highest sensitivity, in terms of increased cytotoxicity, to the both compounds after metabolic activation consistent with the known ability of these two compounds to induce DNA adducts. Strikingly, we found that the RAD54(-/-)/KU70(-/-) cell line, a mutant defective in the repair of double-strand breaks, is sensitive to benzo[a]pyrene, suggesting that this compound also induces strand breaks in these cells. In this study we combined a previously employed method, metabolic activation by S9 mix, with the use of a DNA-repair mutant panel, thereby broadening the range of compounds that can be screened for potential genotoxicity.

Probiotic lactic acid bacteria detoxify N-nitrosodimethylamine.

Humans can be exposed to N-nitroso compounds (NOCs) due to many environmental sources, as well as endogenous formation. The main nitrosamine found in food products and also synthesised in vivo by intestinal microbiota is N-nitrosodimethylamine (NDMA). It can cause cancer of the stomach, kidney and colon. The effect of four probiotic Lactobacillus strains on NDMA was studied under different culture conditions (24 h in MRS, 168 h in modified MRS N, and 168 h in phosphate buffer). HPLC and GC-TEA methods were used for NDMA determination in supernatants. The influence of lactic acid bacteria on NDMA genotoxicity was investigated by means of the comet assay. Additionally, the effect of NDMA (2-100 µg ml⁻¹) on the growth and survival of the probiotic strains was studied. The results indicate that the bacteria decreased NDMA concentration by up to 50%, depending on the culture conditions, time of incubation, NDMA concentration, pH and bacterial strain. Lb. brevis 0945 lowered the concentration and genotoxicity of NDMA most effectively by up to 50%. This could be due to either adsorption or metabolism. The growth and survival of the bacteria was not affected by any of the tested NDMA concentrations.

[Production of nitric oxide metabolites during transplanted tumors growth with different metastatic potential].

The endogenous formation of metabolites of NO - nitrite (NI), nitrates (NA) and volatile nitrosamines in the body, tumor tissue and by abdominal cavity by macrophages for dynamics was investigated in mice F1(C57BlxCBA), Balb/c and BDF with subcutaneous transplanted tumors (Erlich carcinoma - EC and metastatic Lewis lung carcinoma - LLC). It was shown that growth of EC was accompanied by a statistically significant increase in the concentrations of NI and NA in tumor tissue to (7.3±4.67)'10-6 - (7.8±2.57)'10-5 (mol/kg) for the first three weeks and a sharp increase in urinary excretion of NI and NA. The maximum total concentration of NI and NA - (3.,6±0.46)'10-5 in tissue LLC was registered during the early stage of the tumor growth (7 days); it later declined, negatively correlating with the mass of the tumor. NI secretion by abdominal cavity macrophages demonstrated statistically significantly decrease at the stage of intensive growth LLC (14, 21 days). The tissue of EC contained varied concentration of cancerogenic N-nitrosodimethylamine and N-nitrosodiethylamine at all investigated time points. Thus, the ability of different gistogenesis tumor tissue to synthesize metabolites NO depended on time parameters and was more pronounced for EC, than LLC.

Role of aldehydes in the toxic and mutagenic effects of nitrosamines.

α-Hydroxynitrosamine metabolites of nitrosamines decompose to a reactive diazohydroxide and an aldehyde. To test the hypothesis that the aldehydes contribute to the harmful effects of nitrosamines, the toxic and mutagenic activities of three model methylating agents were compared in Chinese hamster ovary cells expressing or not expressing human O6-alkylguanine DNA alkyltransferase (AGT). N-Nitrosomethylurethane (NMUr), acetoxymethylmethylnitrosamine (AMMN), and 4-(methylnitrosamino)-4-acetoxy-1-(3-pyridyl)-1-butanone (NNK-4-OAc) are all activated by ester hydrolysis to methanediazohydroxide. NMUr does not form an aldehyde, whereas AMMN generates formaldehyde, and NNK-4-OAc produces 4-oxo-1-(3-pyridyl)-1-butanone (OPB). Since these compounds were likely to alkylate DNA to different extents, the toxic and mutagenic activities of these compounds were normalized to the levels of the most cytotoxic and mutagenic DNA adduct, O6-mG, to assess if the aldehydes contributed to the toxicological properties of these methylating agents. Levels of 7-mG indicated that the differences in cytotoxic and mutagenic effects of these compounds resulted from differences in their ability to methylate DNA. When normalized against the levels of O6-mG, there was no difference between these three compounds in cells that lacked AGT. However, AMMN and NNK-4-OAc were more toxic than NMUr in cells expressing AGT when normalized against O6-mG levels. In addition, AMMN was more mutagenic than NNK-4-OAc and MNUr in these cells. These findings demonstrate that the aldehyde decomposition products of nitrosamines can contribute to the cytotoxic and/or mutagenic activity of methylating nitrosamines.

Aerobic biotransformation of N-nitrosodimethylamine and N-nitrodimethylamine in methane and benzene amended soil columns.

Aerobic biotransformation of N-nitrosodimethylamine (NDMA), an emerging contaminant of concern, and its structural analog N-nitrodimethylamine (DMN), was evaluated in benzene and methane amended groundwater passed through laboratory scale soil columns. Competitive inhibition models were used to model the kinetics for NDMA and DMN cometabolism accounting for the concurrent degradation of the growth and cometabolic substrates. Transformation capacities for NDMA and DMN with benzene (13 and 23µg (mgcells)(-1)) and methane (0.14 and 8.4µg (mgcells)(-1)) grown cultures, respectively are comparable to those presented in the literature, as were first order endogenous decay rates estimated to be 2.1×10(-2)±1.7×10(-3)d(-1) and 6.5×10(-1)±7.1×10(-1)d(-1) for the methane and benzene amended cultures, respectively. These studies highlight possible attenuation mechanisms and rates for NDMA and DMN biotransformation in aerobic aquifers undergoing active remediation, natural attenuation or managed aquifer recharge with treated wastewater (i.e., reclaimed water).

Ex situ treatment of N-nitrosodimethylamine (NDMA) in groundwater using a fluidized bed reactor.

N-nitrosodimethylamine (NDMA) is a suspected human carcinogen that has traditionally been treated in water using ultraviolet irradiation (UV). The objective of this research was to examine the application of a laboratory-scale fluidized bed reactor (FBR) as an alternative technology for treating NDMA to part-per-trillion (ng/L) concentrations in groundwater. Previous studies have shown that the bacterium Rhodococcus ruber ENV425 is capable of cometabolizing NDMA during growth on propane as a primary substrate in batch culture (Fournier et al., 2009) and in a bench-scale membrane bioreactor (Hatzinger et al., 2011) to low ng/L concentrations. R. ruber ENV425 was inoculated into the FBR during this study. With a hydraulic residence time (HRT) of 20 min, the FBR was found to be an effective means to treat 10-20 µg/L of NDMA to effluent concentrations less than 100 ng/L. When the HRT was increased to 30 min and oxygen and propane addition rates were optimized, the FBR system demonstrated treatment of the NDMA to effluent concentrations of less than 10 ng/L. Short-term shutdowns and the presence of trichloroethene (TCE) at 6 µg/L as a co-contaminant had minimal effect on the treatment of NDMA in the FBR. The data suggest that the FBR technology can be a viable alternative to UV for removing NDMA from groundwater.

Oxidation of methyl and ethyl nitrosamines by cytochrome P450 2E1 and 2B1.

Cytochrome P450 (P450) 2E1 is the major enzyme that oxidizes N-nitrosodimethylamine [N,N-dimethylnitrosamine (DMN)], a carcinogen and also a representative of some nitrosamines formed endogenously. Oxidation of DMN by rat or human P450 2E1 to HCHO showed a high apparent intrinsic kinetic deuterium isotope effect (KIE), ≥8. The KIE was not attenuated in noncompetitive intermolecular experiments with rat liver microsomes {(D)V = 12.5; (D)(V/K) = 10.9 [nomenclature of Northrop, D. B. (1982) Methods Enzymol. 87, 607-625]} but was with purified human P450 2E1 [(D)V = 3.3; (D)(V/K) = 3.7], indicating that C-H bond breaking is partially rate-limiting with human P450 2E1. With N-nitrosodiethylamine [N,N-diethylnitrosamine (DEN)], the intrinsic KIE was slightly lower and was not expressed [e.g., (D)(V/K) = 1.2] in noncompetitive intermolecular experiments. The same general pattern of KIEs was also seen in the (D)(V/K) results with DMN and DEN for the minor products resulting from the denitrosation reactions (CH(3)NH(2), CH(3)CH(2)NH(2), and NO(2)(-)). Experiments with deuterated N-nitroso-N-methyl-N-ethylamine demonstrated that the lower KIEs associated with ethyl versus methyl oxidation could be distinguished within a single molecule. P450 2E1 oxidized DMN and DEN to aldehydes and then to the carboxylic acids. No kinetic lags were observed in acid formation; pulse-chase experiments with carrier aldehydes showed only limited equilibration with P450 2E1-bound aldehydes, indicative of processive reactions, as reported for P450 2A6 [Chowdhury, G., et al. (2010) J. Biol. Chem. 285, 8031-8044]. These same features (no lag phase for HCO(2)H formation and a lack of equilibration in pulse-chase assays) were also seen with (rat) P450 2B1, which has a lower catalytic efficiency for DMN oxidation and a larger active site. Thus, the processivity of dialkyl nitrosamine oxidation appears to be shared by a number of P450s.

Computational evidence for α-nitrosamino radical as initial metabolite for both the P450 dealkylation and denitrosation of carcinogenic nitrosamines.

The mutagenic and carcinogenic potency of α-CH(n)-nitrosamines such as N-nitrosodimethylamine (NDMA) is caused by their P450-catalyzed α-hydroxylation and subsequent dealkylation, yielding alkyl diazonium ions (R-N≡N(+)) as potent electrophiles. Alternatively, P450s may also catalyze their denitrosation as metabolic detoxification. DFT calculations at the UB3LYP/LANL2DZ(Fe)/6-31G+**(H,C,N,O,S)//LANL2DZ(Fe)/6-31G(H,C,N,O,S) level of theory show that H-abstraction from the α-C of NDMA as initial metabolic step yields an α-nitrosamino radical (•CH(2)N(CH(3))NO) as common first intermediate for both the oxidative dealkylation (toxification) and denitrosation (detoxification) pathways. In particular, the calculated kinetic isotope effect for the P450-mediated dealkylation of NDMA is in good agreement with experimental information. The results show further that the initial α-hydroxylation of NDMA may proceed in two spin states. Besides a stepwise high-spin (HS, quartet) route with a separate rebound barrier, there is a concerted low-spin (LS, doublet) pathway. Interestingly, the resultant two-state reactivity appears to discriminate between metabolic toxification and detoxification: Evaluation of calculated free energy barriers of the H-abstraction (ΔG(‡)) through the Eyring equation suggests that the dealkylation:denitrosation product ratio is governed by the LS:HS ratio of the overall metabolic process. Moreover, inclusion of three further α-CH(n)-nitrosamines in the computational analysis demonstrates that the initial H-abstraction barrier is proportional to the C-H bond dissociation enthalpy (BDE) of the substrates, which enables the estimation of spin-averaged reaction barriers through ground-state BDE calculations. The discussion includes also reductive denitrosation pathways that according to current computational evidence appear to be unlikely for aliphatic nitrosamines.

Acute effects of ethanol on the transfer of nicotine and two dietary carcinogens in human placental perfusion.

Many mothers use, against instructions, alcohol during pregnancy. Simultaneously mothers are exposed to a wide range of other environmental chemicals. These chemicals may also harm the developing fetus, because almost all toxic compounds can go through human placenta. Toxicokinetic effects of ethanol on the transfer of other environmental compounds through human placenta have not been studied before. It is known that ethanol has lytic properties and increases the permeability and fluidity of cell membranes. We studied the effects of ethanol on the transfer of three different environmental toxins: nicotine, PhIP (2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine) and NDMA (N-nitrosodimethylamine) in placental perfusion. We tested in human breast cancer adenocarcinoma cell line MCF-7 whether ethanol affects ABCG2/BCRP, which is also the major transporter in human placenta. We found that the transfer of ethanol is comparable to that of antipyrine, which points to passive diffusion as the transfer mechanism. Unexpectedly, ethanol had no statistically significant effect on the transfer of the other studied compounds. Neither did ethanol inhibit the function of ABCG2/BCRP. These experiments represent only the effects of acute exposure to ethanol and chronic exposure remains to be studied.

N-Nitroso compounds and cancer incidence: the European Prospective Investigation into Cancer and Nutrition (EPIC)-Norfolk Study.

BACKGROUND: Humans are exposed to preformed N-nitroso compounds (NOCs) and endogenous NOCs. Several NOCs are potential human carcinogens, including N-nitrosodimethylamine (NDMA), but evidence from population studies is inconsistent. OBJECTIVE: We examined the relation between dietary NOCs (NDMA), the endogenous NOC index, and dietary nitrite and cancer incidence in the European Prospective Investigation into Cancer and Nutrition (EPIC)-Norfolk, United Kingdom, study. DESIGN: This was a prospective study of 23,363 men and women, aged 40-79 y, who were recruited in 1993-1997 and followed up to 2008. The baseline diet was assessed with food-frequency questionnaires. RESULTS: There were 3268 incident cancers after a mean follow-up of 11.4 y. Dietary NDMA intake was significantly associated with increased cancer risk in men and women [hazard ratio (HR): 1.14; 95% CI: 1.03, 1.27; P for trend = 0.03] and in men (HR: 1.24; 95% CI: 1.07, 1.44; P for trend = 0.005) when the highest quartile was compared with the lowest quartile in age- and sex-adjusted analyses but not in multivariate analyses (HR: 1.10; 95% CI: 0.97, 1.24; HR for men: 1.18; 95% CI: 1.00, 1.40; P for trend ≥ 0.05). When continuously analyzed, NDMA was associated with increased risk of gastrointestinal cancers (HR: 1.13; 95% CI: 1.00, 1.28), specifically of rectal cancer (HR: 1.46; 95% CI: 1.16, 1.84) per 1-SD increase after adjustment for age, sex, body mass index, cigarette smoking status, alcohol intake, energy intake, physical activity, education, and menopausal status (in women). The endogenous NOC index and dietary nitrite were not significantly associated with cancer risk. There was a significant interaction between plasma vitamin C concentrations and dietary NDMA intake on cancer incidence (P for interaction < 0.00001). CONCLUSIONS: Dietary NOC (NDMA) was associated with a higher gastrointestinal cancer incidence, specifically of rectal cancer. Plasma vitamin C may modify the relation between NDMA exposure and cancer risk.

N-acetylcysteine attenuates dimethylnitrosamine induced oxidative stress in rats.

Oxidative stress has been implicated in the pathogenesis and progression of various hepatic disorders and hence screening for a good hepatoprotective and antioxidant agent is the need of the hour. The present study was aimed to investigate the hepatoprotective and antioxidant property of N-acetylcysteine (NAC) against dimethylnitrosamine (DMN) induced oxidative stress and hepatocellular damage in male Wistar albino rats. Administration of single dose of DMN (5mg/kg b.w.; i.p.) resulted in significant elevation in the levels of serum aspartate transaminase and alanine transaminase, indicating hepatocellular damage. Oxidative stress induced by DMN treatment was confirmed by an elevation in the status of lipid peroxidation (LPO) and reduction in the activities of enzymic antioxidants such as superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione-S-transferase and in the levels of non-enzymic antioxidants, reduced glutathione, vitamin-C and vitamin-E in the liver tissue. DMN induced oxidative stress and hepatocellular membrane instability was further substantiated by a decline in the status of the membrane bound ATPases in the liver tissue. Post-treatment with NAC (50mg/kg b.w.; p.o.) for 7days effectively protected against the DMN induced insult to liver by preventing the elevation in the status of the serum marker enzymes and LPO, and restoring the activities of both the enzymic and non-enzymic antioxidants and membrane bound ATPases towards normalcy. These results demonstrate that NAC acts as a good hepatoprotective and antioxidant agent in attenuating DMN induced oxidative stress and hepatocellular damage.

The binding affinity of carcinogenic N-nitrosodimethylamine and N-nitrosomethylaniline to cytochromes P450 2B4, 2E1 and 3A6 does not dictate the rate of their enzymatic N-demethylation.

The interaction of carcinogenic N-nitrosodimethylamine (NDMA) and N-nitrosomethylaniline (NMA) with cytochromes P450 (CYP), CYP2B4, CYP2E1 and CYP3A6, and their metabolism by these enzymes reconstituted with NADPH-CYP reductase in liposomes were studied. Using the difference spectroscopy, the lowest values of spectral dissociation constants (KS) of the binary complex of NDMA and NMA with the enzyme were found for CYP2E1. Both N-nitrosamines bind to the heme iron atom as ligands. On the contrary, the binding of NDMA and NMA to CYP2B4 gives the type I spectra. NDMA is bound to CYP3A6 analogously as to CYP2B4, whereas no difference spectra were acquired with NMA and CYP3A6. All tested CYPs oxidize NDMA and NMA. CYP2E1 exhibits the lowest Km values, 7.5 and 5.0 micromol/l for NDMA and NMA, respectively, and for CYP3A6 we found 30.0 and 10.0 micromol/l for NDMA and NMA, respectively, while CYP2B4 exhibits the lowest affinity for both carcinogens. In spite of different binding affinities of NDMA and NMA, the values of Vmax for their oxidation were, however, similar for all tested CYPs. The results demonstrate that investigations utilizing several enzymatic approaches are necessary to properly evaluate the mechanism and efficiency of NDMA and NMA oxidation by CYPs in vitro.