Removal of N-nitrosopyrrolidine from GAC by a three-dimensional electrochemical reactor: degradation mechanism and degradation path.

Nitrogen-containing disinfection by-products (N-DBPs) produced in the process of drinking water disinfection are widely concerning due to the high cytotoxicity and genotoxicity. It is due to the difficulty of natural degradation of N-DBPs in water and the fact that conventional treatment systems do not effectively treat N-DBPs in drinking water. In this study, N-nitrosopyrrolidine (NPYR) in water was electrocatalytically degraded by a three-dimensional electrode reactor (3DER). This system applied graphite plates as anode and cathode. The granular activated carbon (GAC) was used as third electrode. The degradation of NPYR using a continuous flow three-dimensional electrode reactor was investigated by examining the effects of flow rate, current density, electrolyte concentration, and pollutant concentration on the degradation efficiency, energy consumption, and reaction kinetics of GAC particle electrodes. The results showed that the optimal operating conditions were flow rate = 0.45 mL/min, current density = 6 mA/cm2, Na2SO4 concentration = 0.28 mol/L, and NPYR concentration = 20 mg/L. Under optimal conditions, the degradation of NPYR exceeded 58.84%. The main contributor of indirect oxidation was deduced from free radical quenching experiments. NPYR concentration was measured by GC-MS with DB-5 capillary column, operating in full scan monitoring mode for appropriate quantification of NPYR and intermediates. Based on the identification of reaction intermediates, a possible pathway for the electrochemical oxidation of NPYR on GAC particle electrodes was proposed.

Efficient degradation of N-nitrosopyrrolidine using CoFe-LDH/AC particle electrode via heterogeneous Fenton-like reaction.

With the rapid development of drinking water disinfection technology, extensive attentions are paid to the nitrogenous disinfection by-products (N-DBPs) that has strong carcinogenicity, thus their degradation becomes important for the health of human beings. In this work, for the first time, CoFe-LDH material used as particle electrode is proposed to treat trace N-nitrosopyrrolidine (NPYR) in a three-dimensional aeration electrocatalysis reactor (3DAER). The factors on the degradation efficiency and energy consumption of NPYR are systematically investigated, and the results of radical quenching experiments show that the degradation of NPYR is completed by combining with ·OH, ·O2and direct oxidation together. CoFe-LDH particle electrode plays a vital role in generating ·OH via heterogeneous ‾Fenton-like reaction. Moreover, the adsorbed saturated CoFe-LDH particle electrode can be regenerated by electrochemical action to induce further recycle adsorption and form in-situ electrocatalysis. This work pave a way for the removal of NPYR with high efficiency, low energy conservation and environmental protection.

Long-Term Stability of Volatile Nitrosamines in Human Urine.

Volatile nitrosamines (VNAs) are established teratogens and carcinogens in animals and classified as probable (group 2A) and possible (group 2B) carcinogens in humans by the IARC. High levels of VNAs have been detected in tobacco products and in both mainstream and sidestream smoke. VNA exposure may lead to lipid peroxidation and oxidative stress (e.g., inflammation), chronic diseases (e.g., diabetes) and neurodegenerative diseases (e.g., Alzheimer's disease). To conduct epidemiological studies on the effects of VNA exposure, short-term and long-term stabilities of VNAs in the urine matrix are needed. In this report, the stability of six VNAs (N-nitrosodimethylamine, N-nitrosomethylethylamine, N-nitrosodiethylamine, N-nitrosopiperidine, N-nitrosopyrrolidine and N-nitrosomorpholine) in human urine is analyzed for the first time using in vitro blank urine pools fortified with a standard mixture of all six VNAs. Over a 24-day period, analytes were monitored in samples stored at ∼20°C (collection temperature), 4-10°C (transit temperature) and -20 and -70°C (long-term storage temperatures). All six analytes were stable for 24 days at all temperatures (n = 15). The analytes were then analyzed over a longer time period at -70°C; all analytes were stable for up to 1 year (n = 62). A subset of 44 samples was prepared as a single batch and stored at -20°C, the temperature at which prepared samples are stored. These prepared samples were run in duplicate weekly over 10 weeks, and all six analytes were stable over the entire period (n = 22).

Carcinogenic nitrosamines in traditional beer as the cause of oesophageal squamous cell carcinoma in black South Africans.

BACKGROUND: Before the 1930s, squamous cell carcinoma (SCC) of the oesophagus was almost unknown among black South Africans. From the 1930s the annual frequency rose. A dietary cause was sought, the staple diet of black people having changed from sorghum to maize (corn), with traditional beer being brewed from maize. Carcinogenic N-nitrosamines in traditional beer were suggested as a cause of SCC of the oesophagus, with Fusarium moniliforme, a corn saprophyte, thought to play a role. OBJECTIVES: To confirm the presence of N-nitrosamines in traditional beer and demonstrate a mechanism for the oncogenesis of oesophageal carcinoma. METHODS: Analysis by high-performance liquid chromatography was conducted for the identification of nitrosamines in traditional beer samples, and molecular docking studies were employed to predict the affinity between N-nitrosamines and the S100A2 protein. RESULTS: Carcinogenic N-nitrosamines were identified in all six samples of traditional beer examined (N=18 analyses), and docking studies confirmed a high affinity of the nitrosamine N-nitrosopyrrolidone with the S100A2 protein. This may result in the altered expression of the S100A2 protein, leading to tumour progression and prognosis. CONCLUSION: It is suggested that carcinogenic N-nitrosamines in traditional beer are a major factor in the causation of SCC of the oesophagus in black South Africans. N-nitrosamines have been shown to produce cancer experimentally, but there has not been conclusive epidemiological evidence that N-nitrosamines are carcinogenic to humans. This study is the first to demonstrate the potential link between N-nitrosamines and a human tumour.

Evaluation of repeated dose micronucleus assays of the liver using N-nitrosopyrrolidine: a report of the collaborative study by CSGMT/JEMS.MMS.

The repeated dose liver micronucleus (RDLMN) assay has the potential to detect liver carcinogens, and can be integrated into a general toxicological study. To assess the performance of the assay, N-nitrosopyrrolidine (NPYR), a genotoxic hepatocarcinogen, was tested in 14- or 28-day RDLMN assays. NPYR was orally administered to rats at a daily dose of 25, 50 or 100 mg/kg. One day after the last administration, a portion of the liver was removed and hepatocyte micronucleus (MN) specimens were prepared by the new method recently established by Narumi et al. In addition, a bone marrow MN assay and a histopathological examination of the liver were conducted. The detection of Phospho-Histone H3 was performed by immunohistochemistry to evaluate the proliferation rate of hepatocytes. The results showed significant increase in the number of micronucleated hepatocytes and Phospho-Histone H3-positive cells from the lowest dose in both 14- and 28-day RDLMN assays. On the other hand, the bone marrow MN assay yielded a negative result, which was in accordance with the existing report of the bone marrow MN assay using mice. Upon histopathological examination, inflammatory lesions and hypertrophy were noted, which may explain the increase in the hepatocyte proliferation and the enhancement of MN induction by NPYR. Our findings indicate that the RDLMN assay could be a useful tool for comprehensive risk assessment of carcinogenicity by providing information on both genotoxicity and histopathology when integrated into a general repeat dosing toxicity assay.

Inhibitors of differentiation-1 promotes nitrosopyrrolidine-induced transformation of HPV 16-immortalized cervical epithelial cell.

Our previous study implied a correlation between inhibitors of differentiation-1 (Id-1) and cervical cancer development. However, how Id-1 contributes to cervical carcinogenesis is unknown. In the present study, we used an in vitro transformation model to investigate the role of Id-1 in the transformation of cervical cells. Human papillomavirus (HPV)-immortalized cervical epithelial cells (H8) were successfully transformed by exposure to the carcinogen N-nitrosopyrrolidine (NPYR). The expression of both Id-1 RNA and protein was significantly increased in transformed H8 cells, suggesting a possible role of Id-1 in cervical cell transformation. Ectopic expression of Id-1 in H8 cells potentiated NPYR-induced cell transformation. In contrast, silencing of Id-1 suppressed NPYR-induced H8 cell transformation. In addition, the expression of HPV E6 and E7 oncoproteins was upregulated while that of the tumor suppressors p53 and pRb was suppressed after H8 cell transformation. Our results suggest that Id-1 plays an oncogenic role in HPV-related cervical carcinogenesis, which sheds light on cervical cancer development mechanisms and implies that Id-1 is a potential target for cervical cancer prevention and therapy.

Quantitation of urinary volatile nitrosamines from exposure to tobacco smoke.

A sensitive and selective method was developed and validated to detect six volatile nitrosamines (N-nitrosodimethylamine, N-nitrosomethylethylamine, N-nitrosodiethylamine, N-nitrosopiperidine, N-nitrosopyrrolidine and N-nitrosomorpholine) in human urine. This method uses a liquid-liquid extraction cartridge followed by analysis with gas chromatography-tandem mass spectrometry (GC-MS-MS) and quantification based on isotopic dilution. This is the first GC-MS-MS method reported for measuring volatile nitrosamines in human urine. This method reduces the sample volume required in other methods from 5-25 to 2 mL. The limits of detection (2.62, 1.99, 2.73, 0.65, 0.25, 3.66 pg/mL, respectively) were better than existing methods, largely because of improved positive chemical ionization achieved by using ammonia gas and reducing background noise. Using nitrogen as the collision gas allowed the confirmation transition in the low mass region to be monitored. The analysis of human urine using this validated method is accurate (relative bias of 0-19%) and precise (relative standard deviation of 0.2-18% over two months of analyses). The validated method was applied to 100 urine samples and the levels of all six volatile nitrosamines were reported for the first time in urine specimens collected from smokers and nonsmokers, with smoking status determined by urinary cotinine measurement. Among 100 smokers and nonsmokers, the levels of three analytes (N-nitrosodimethylamine, N-nitrosomethylethylamine and N-nitrosopiperidine) were significantly higher in smokers than nonsmokers (p < 0.05).

Spanish honeys protect against food mutagen-induced DNA damage.

BACKGROUND: Honey contains a variety of polyphenols and represents a good source of antioxidants, while the human diet often contains compounds that can cause DNA damage. The present study investigated the protective effect of three commercial honey samples of different floral origin (rosemary, heather and heterofloral) from Madrid Autonomic Community (Spain) as well as an artificial honey on DNA damage induced by dietary mutagens, using a human hepatoma cell line (HepG2) as in vitro model system and evaluation by the alkaline single-cell gel electrophoresis or comet assay. RESULTS: Rosemary, heather and heterofloral honeys protected against DNA strand breaks induced by N-nitrosopyrrolidine (NPYR), benzo(a)pyrene (BaP) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), but none of the honey samples tested prevented DNA strand breaks induced by N-nitrosodimethylamine (NDMA). Heterofloral and heather (unifloral) honeys with higher phenolic content were most effective in protecting HepG2 cells against DNA damage induced by food mutagens. Heterofloral honey was more protective against NPYR and BaP, while heather honey was more protective against PhIP. Artificial honey did not show a protective effect against DNA damage induced by any of the food mutagens tested, indicating that the protective effects of honeys could not be due to their sugar components. CONCLUSION: The results suggest that the protective effect of three kinds of Spanish honey of different floral origin could be attributed in part to the phenolics present in the samples. Honeys with higher phenolic content, i.e. heather and heterofloral honeys, were most effective in protecting against food mutagen-induced DNA damage in HepG2 cells. In addition, a possible synergistic effect between other minor honey components could also be involved.

The enhancing effect of ethanol on the mutagenic activation of N-nitrosomethylbenzylamine by cytochrome P450 2A in the rat oesophagus.

Alcohol consumption is frequently associated with various cancers and the enhancement of the metabolic activation of carcinogens has been proposed as a mechanism underlying this relationship. The ethanol-induced enhancement of N-nitrosodiethylamine (DEN)-mediated carcinogenesis can be attributed to an increase in hepatic activity. However, the mechanism of elevation of N-nitrosomethylbenzylamine (NMBA)-induced tumorigenesis remains unclear. To elucidate the mechanism underlying the role of ethanol in the enhancement of NMBA-induced oesophageal carcinogenesis, we evaluated the hepatic and extrahepatic levels of the cytochrome P450 (CYP) and mutagenic activation of environmental carcinogens by immunoblot analyses and Ames preincubation test, respectively, in F344 rats treated with ethanol. Five weeks of treatment with 10% ethanol added to the drinking water or two intragastric treatments with 50% ethanol, both resulted in elevated levels of CYP2E1 (1.5- to 2.3-fold) and mutagenic activities of DEN, N-nitrosodimethylamine and N-nitrosopyrrolidine in the presence of rat liver S9 (1.5- to 2.4-fold). This was not the case with CYP1A1/2, CYP2A1/2, CYP2B1/2 or CYP3A2, nor with the activities of 2-amino-3-methylimidazo[4,5-f]quinoline, 3-amino-1-methyl-5H-pyrido[4,3-b]indole, aflatoxin B(1) or other N-nitroso compounds (NOCs), including NMBA. Ethanol-induced elevations of CYP2A and CYP2E1 were observed in the oesophagus (up to 1.7- and 2.3-fold) and kidney (up to 1.5- and 1.8-fold), but not in the lung or colon. In oesophagus and kidney, the mutagenic activities of NMBA and four NOCs were markedly increased (1.3- to 2.4-fold) in treated rats. The application of several CYP inhibitors revealed that CYP2A were likely to contribute to the enhancing effect of ethanol on NMBA activation in the rat oesophagus and kidney, but that CYP2E1 failed to do so. These results showed that the enhancing effect of ethanol on NMBA-induced oesophageal carcinogenesis could be attributed to an increase in the metabolic activation of NMBA by oesophageal CYP2A during the initiation phase, and that this occurred independently of CYP2E1.

Evaluation of the in vitro antimutagenic effect of Doash tea aqueous extracts.

Human carcinogens are formed mainly due to the lifestyle and diet that is followed. It is well known that dietary factors play a crucial role in the aetiology of human cancer. The new attractions of drug discovery using natural products remain an important issue in the current herbal medicine research. The present study aimed to evaluate the antimutagenic activity of the water extracts of Doash leaves against several known mutagens, both direct- and indirect-acting, belonging to different chemical classes. These classes are heterocyclic amines (HAs), polycyclic aromatic hydrocarbons and nitrosamines. The antimutagenic activity will be determined in Salmonella/microsomal system (Ames) using strains of Salmonella Typhimurium. Four Salmonella bacterial strains (TA98, TA97, TA100 and TA1530) were used in the present study. Results obtained showed that Doash extract possesses powerful antimutagenic properties, which impair the deleterious effects of various chemicals used in this study. One possible mechanism involved in this protection is the inhibition of the metabolic activation of chemical carcinogens to their reactive metabolites. We also suggest that the health benefits of Doash could be derived from the additive and synergistic combinations of the various phytochemicals present in Doash leaves. Other studies should also be conducted to determine the active components of Doash leaves, including macronutrients, micronutrients and other phytochemicals. Clinical studies should be performed before any claims that Doash consumption offers chemoprotection against cancer can be made.

Time-series analysis of gene expression profiles induced by nitrosamides and nitrosamines elucidates modes of action underlying their genotoxicity in human colon cells.

N-nitroso compounds (NOCs) may represent a carcinogenic risk to humans following endogenous colonic nitrosation processes. We used the colon adenocarcinoma cell line Caco-2 to investigate transcriptomic changes at three time points (1, 6, 24 h) following exposure to genotoxic concentrations of six different NOCs (two nitrosamides, four nitrosamines) with the purpose of identifying biological processes that may play a part in the carcinogenicity of these compounds. This is especially important for nitrosamide exposure where, in light of their high reactivity, important gene expression modifications may take place early in the exposure. We also analyzed NOC-induced O(6)-methylguanine adducts in relation to transcriptomics since these adducts may influence the expression of genes pivotal in NOC-associated carcinogenicity. Many modified pathways appeared related to DNA damage, cell cycle, apoptosis, growth factor signaling and differentiation, which are linked with carcinogenicity. Nitrosamides showed the strongest response at 1h of exposure, while nitrosamines had the strongest effect at 6 and 24 h. Additionally, methylation was strongly associated with processes that may contribute to the carcinogenic risk. In summary, we have found that NOC-induced gene expression changes vary over time and that many of the modified pathways and processes indicate a carcinogenic risk associated with NOC exposure.

Evolution of research on the DNA adduct chemistry of N-nitrosopyrrolidine and related aldehydes.

This perspective reviews our work on the identification of DNA adducts of N-nitrosopyrrolidine and some related aldehydes. The research began as a focused project to investigate mechanisms of cyclic nitrosamine carcinogenesis but expanded into other areas, as aldehyde metabolites of NPYR were shown to have their own diverse DNA adduct chemistry. A total of 69 structurally distinct DNA adducts were identified, and some of these, found in human tissues, have provided intriguing leads for investigating carcinogenesis mechanisms in humans due to exposure to both endogenous and exogenous agents.

Adsorption of nitrosamines by mesoporous zeolite.

On the basis of a study of the adsorption of zeolite and mesoporous silica, we attempted to create a hierarchical structure in the new nitrosamines trapper. Thus, mesoporous HZSM-5 zeolite was fabricated through impregnating a structure-directing agent into the as-synthesized MCM-41 followed by dry-gel conversion to transform amorphous silica to zeolite crystal. The texture of mesoporous ZSM-5 was tailored by adjusting the Si/Al ratio in the MCM-41 source and the thermal treatment time. The resulting samples were characterized by N(2) adsorption to evaluate their textural properties. One volatile nitrosamine, N-nitrosopyrrolidine (NPYR), was used as probe molecule in instantaneous adsorption to survey the function of the resulting composites. Adsorptions of N'-nitrosonornicotine (NNN) in dichloromethane solution and tobacco-specific nitrosamines (TSNA) in tobacco-extract solution were also utilized for the same purpose. As expected, mesoporous zeolite exhibits a good adsorption capacity in laboratory tests, superior to either microporous zeolite or mesoporous silica, providing a valuable candidate for controlling nitrosamines in the environment.

Evaluation of the influence of proline, hydroxyproline or pyrrolidine in the presence of sodium nitrite on N-nitrosamine formation when heating cured meat.

N-nitrosamines are meant to be probable or possible carcinogenic components, possibly formed out of a reaction between nitrite and N-containing substances such as amino acids and secondary amines. Nitrite is often used for processing meat products because of its colouring and antimicrobial properties. During this experimental setup, the influence of proline, hydroxyproline or pyrrolidine on N-nitrosamine formation in meat samples was evaluated. The N-nitrosamines concentrations were measured with gas chromatography-thermal energy analyzer. Only the concentrations of N-nitrosodimethylamine and N-nitrosopyrrolidine were found above the limit of detection in a number of tested experimental conditions. The concentration of these two N-nitrosamines was modelled as a function of temperature and nitrite concentration for different situations (presence or absence of added natural N-containing meat components). It could be concluded that proline and pyrrolidine promoted the formation of N-nitrosopyrrolidine. It could also be confirmed that the higher the temperature of the meat processing procedure and the higher the sodium nitrite amounts added, the higher were the yields of the respective N-nitrosamines.

Effective nitrosamines trap derived from the in situ carbonized mesoporous silica MCM-41.

A new route to modify the mesoporous silica MCM-41 with carbon, using the inherent surfactant template in the as-synthesized sample as the carbon precursor, is reported in this article. Apart from the advantage of omitting energy and time required for removal of template, the resulting silica-carbon composites exhibit a high efficiency in adsorption and catalytic decomposition of N-nitrosopyrrolidine (NPYR), the typical carcinogenic pollutant in environment. The influence of carbonization temperature on the structure and performance of the resulting carbon-silica composite was investigated by use of XRD, N(2) adsorption-desorption, FTIR and TG-DSC techniques, and the instantaneous adsorption and the temperature-programmed surface reaction (TPSR) of NPYR as well as the reduction of nitrosamines level of cigarette smoke were also utilized to assess the actual function of these composites. The carbonization performed in the range of 773-973 K enabled the resulting composites to possess a higher activity than parent MCM-41 in eliminating volatile nitrosamines, which is beneficial for controlling carcinogenic pollutants in environment.

Mass spectrometric analysis of a cyclic 7,8-butanoguanine adduct of N-nitrosopyrrolidine: comparison to other N-nitrosopyrrolidine adducts in rat hepatic DNA.

The well established rat hepatocarcinogen N-nitrosopyrrolidine (NPYR, 1) requires metabolic activation to DNA adducts to express its carcinogenic activity. Among the NPYR-DNA adducts that have been identified, the cyclic 7,8-butanoguanine adduct 2-amino-6,7,8,9-tetrahydro-9-hydroxypyrido[2,1-f]purine-4(3H)-one (6) has been quantified using moderately sensitive methods, but its levels have never been compared to those of other DNA adducts of NPYR in rat hepatic DNA. Therefore, in this study, we developed a sensitive new LC-ESI-MS/MS-SRM method for the quantitation of adduct 6 and compared its levels to those of several other NPYR-DNA adducts formed by different mechanisms. The new method was shown to be accurate and precise, with good recoveries and low fmol detection limits. Rats were treated with NPYR by gavage at doses of 46, 92, or 184 mg/kg body weight and sacrificed 16 h later. Hepatic DNA was isolated and analyzed for NPYR-DNA adducts. Adduct 6 was by far the most prevalent, with levels ranging from about 900-3000 micromol/mol Gua and responsive to dose. Levels of adducts formed from crotonaldehyde, a metabolite of NPYR, were about 0.2-0.9 micromol/mol dGuo, while those of adducts resulting from reaction with DNA of tetrahydrofuranyl-like intermediates were in the range of 0.01-4 micromol/mol deoxyribonucleoside. The results of this study demonstrate that, among typical NPYR-DNA adducts, adduct 6 is easily the most abundant in hepatic DNA. Since previous studies have shown that it can be detected in the urine of NPYR-treated rats, the results suggest that it is a potential candidate as a biomarker for assessing human exposure to and metabolic activation of NPYR.

A cell-microelectronic sensing technique for profiling cytotoxicity of chemicals.

A cell-microelectronic sensing technique is developed for profiling chemical cytotoxicity and is used to study different cytotoxic effects of the same class chemicals using nitrosamines as examples. This technique uses three human cell lines (T24 bladder, HepG2 liver, and A549 lung carcinoma cells) and Chinese hamster ovary (CHO-K1) cells in parallel as the living components of the sensors of a real-time cell electronic sensing (RT-CES) method for dynamic monitoring of chemical toxicity. The RT-CES technique measures changes in the impedance of individual microelectronic wells that is correlated linearly with changes in cell numbers during t log phase of cell growth, thus allowing determination of cytotoxicity. Four nitrosamines, N-nitrosodimethylamine (NDMA), N-nitrosodiphenylamine (NDPhA), N-nitrosopiperidine (NPip), and N-nitrosopyrrolidine (NPyr), were examined and unique cytotoxicity profiles were detected for each nitrosamine. In vitro cytotoxicity values (IC(50)) for NDPhA (ranging from 0.6 to 1.9 mM) were significantly lower than the IC(50) values for the well-known carcinogen NDMA (15-95 mM) in all four cell lines. T24 cells were the most sensitive to nitrosamine exposure among the four cell lines tested (T24>CHO>A549>HepG2), suggesting that T24 may serve as a new sensitive model for cytotoxicity screening. Cell staining results confirmed that administration of the IC(50) concentration from the RT-CES experiments inhibited cell growth by 50% compared to the controls, indicating that the RT-CES method provides reliable measures of IC(50). Staining and cell-cycle analysis confirmed that NDPhA caused cell-cycle arrest at the G0/G1 phase, whereas NDMA did not disrupt the cell cycle but induced cell death, thus explaining the different cytotoxicity profiles detected by the RT-CES method. The parallel cytotoxicity profiling of nitrosamines on the four cell lines by the RT-CES method led to the discovery of the unique cytotoxicity of NDPhA causing cell-cycle arrest. This study demonstrates a new approach to comprehensive testing of chemical toxicity.

Inhibition by vitamin C of apoptosis induced by N-nitrosamines in HepG2 and HL-60 cells.

The aim of this study was to evaluate the effect of vitamin C towards N-nitrosopyrrolidine (NPYR)- and N-nitrosodimethylamine (NDMA)-induced apoptosis in human hepatoma (HepG2) and leukemia (HL-60) cell lines using flow cytometry analysis and the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assay (TUNEL). None of the vitamin C concentrations tested (1-100 microM) caused cytotoxicity in HepG2 cells. However, there were significant losses of HL-60 cells viability, measured by MTT assay, 72 h after treatment with 50 and 100 microM vitamin C (29 and 46%, respectively). Moreover, an increase of lactate dehydrogenase release was significant with 50 microM at 72 h (28%) and with 100 microM of vitamin C at 48 and 72 h (27 and 36%, respectively). Also, the percentage of apoptotic HL-60 cells found in TUNEL assay increased to 21% when they were treated with 100 microM vitamin C for 72 h. Thus, in subsequent simultaneous treatments with NPYR (30 and 50 mM) or NDMA (27 and 68 mM) and vitamin C, concentrations of 5-50 microM vitamin C were used. Our results revealed that vitamin C, at all concentrations and times tested, reduced the apoptosis induced by NPYR and NDMA in both cell lines, showing a similar effect in HepG2 and HL-60 cells towards NPYR (50 mM)--65 and 63% of reduction, respectively--whereas towards NDMA (27 mM) the inhibition was higher in HL-60 than in HepG2 cells--75 and 57%, respectively. Therefore, our findings suggest that inhibition of apoptosis may be one of the mechanisms by which vitamin C exerts its protective effect.

Protective effect of vitamin C towards N-nitrosamine-induced DNA damage in the single-cell gel electrophoresis (SCGE)/HepG2 assay.

The aim of this study was to investigate the protective effect of vitamin C towards N-nitrosamine-induced DNA damage in the single-cell gel electrophoresis (SCGE)/HepG2 assay. None of the vitamin C concentrations tested (1-10 microM) in presence or absence of formamidopyrimidine-DNA glycosylase (Fpg enzyme) caused DNA damage per se. HepG2 cells simultaneously treated with vitamin C and N-nitrosodimethylamine (NDMA), N-nitrosopyrrolidine (NPYR), N-nitrosodibutylamine (NDBA) or N-nitrosopiperidine (NPIP) reduced the genotoxic effects of the N-nitrosamines in a dose-dependent manner. At concentrations of 1-5 microM vitamin C, the protective effect was higher towards NPYR-induced oxidative DNA damage (78-79%) than against NDMA (39-55%), NDBA (12-14%) and NPIP (3-55%), in presence of Fpg enzyme. However, a concentration of 10 microM vitamin C led to a maximum reduction in NDBA (94%), NPYR (81%), NPIP (80%) and NDMA (61%)-induced oxidative DNA damage, in presence of Fpg enzyme. The greatest protective effect of vitamin C (10 microM) was higher towards NDBA-induced oxidative DNA damage. One feasible mechanism by which vitamin C exerted its protective effect is that may interact with the enzyme systems catalyzing the metabolic activation of the N-nitrosamines, blocking the production of genotoxic intermediates. Vitamin C (10 microM) strongly reduced the coumarin hydroxylase (82%) activity. However, the p-nitrophenol hydroxylase and the ethoxyresorufine O-deethylation activities were slightly and weakly reduced (32-19%), respectively.