Nitrosomorpholine Research Articles

Use of the benchmark-dose (BMD) approach to derive occupational exposure limits (OELs) for genotoxic carcinogens: N-nitrosamines.

N-Nitrosamines are potent carcinogens and considered non-threshold carcinogens in various regulatory domains. However, recent data indicate the existence of a threshold for genotoxicity, which can be adequately demonstrated. This aspect has a critical impact on selecting the methodology that is applied to derive occupational exposure limits (OELs). OELs are used to protect workers potentially exposed to various chemicals by supporting the selection of appropriate control measures and ultimately reducing the risk of occupational cancer. Occupational exposures to nitrosamines occur during manufacturing processes, mainly in the rubber and chemical industry. The present study derives OELs for inhaled N-nitrosamines, employing the benchmark dose (BMD) approach if data are adequate and read-across for nitrosamines without adequate data. Additionally, benchmark dose lower confidence limit (BMDL) is preferred and more suitable point-of-departure (PoD) to calculate human health guidance values, including OEL. The lowest OEL (0.2μg/m3 ) was derived for nitrosodiethylamine (NDEA), and nitrosopiperidine (NPIP) (OEL = 0.2μg/m3 ), followed by nitrosopyrrolidine (NPYR) (0.4μg/m3 ), nitrosodimethylamine (NDMA), nitrosodimethylamine (NMEA), and nitrosodipropylamine (NDPA) (0.5μg/m3 ), nitrosomorpholine (NMOR) (OEL = 1μg/m3 ), and nitrosodibutylamine (NDBA) (OEL = 2.5μg/m3 ). Limits based on "non-threshold" TD50 slope calculation were within a 10-fold range. These proposed OELs do not consider skin absorption of nitrosamines, which is also a possible route of entry into the body, nor oral or other environmental sources. Furthermore, we recommend setting a limit for total nitrosamines based on the occupational exposure scenario and potency of components.

Possible ways to minimize nitrosation reactions during post-combustion CO2 capture process

The formation and any emission of nitrosamines in a post-combustion CO2 capture process are of great interest over the past few years even though its concentration is quite low. In the present research, bench scale nitrosation experiments were conducted using morpholine, a representative of secondary amines, with NO2 gas and flue gas. Laboratory experiments with 5M morpholine with 100ppm NO2 gas resulted in 88μg/g nitrosomorpholine (NSMO) in 6h and 450μg/g in 24h. However, coal derived flue gas gave only about 7μg/g NSMO in 24h. To prove the inhibitory effect from SO2 in the flue gas, experiments were conducted with diluted NO2 gas blended with SO2 gas in the feed stream. Formation of NSMO was inhibited by about 53.3% when equal concentrations of SO2 and NO2 existed compared with the same concentration of NO2 gas diluted with N2. The inhibitory effect of SO2 gas was found to be linear with its concentration. Apart from that, the addition of various inhibitors to the amine solution was also evaluated and isoascorbic acid was found the best with 86% inhibition activity under the test conditions. Isoascorbic acid was further examined both under simulated CO2 scrubber and stripper conditions indicating good stability and inhibition performance.

Effect of toll-like receptor 7 and 9 targeted therapy to prevent the development of hepatocellular carcinoma.

Chronic liver disease is a predisposing factor for development of hepatocellular carcinoma (HCC). Toll-like receptors play a crucial role in immunity against microbial pathogens and recent evidence suggests that they may also be important in pathogenesis of chronic liver disease. The purpose of this study was to determine whether TLR7 and TLR9 are potential targets for prevention and progression of HCC. Tissue microarrays containing liver samples from patients with cirrhosis, viral hepatitis and HCC were examined for expression of TLR7 and TLR9 and the data obtained was validated in liver specimens from the hospital archives. Proliferation of human HCC cell lines was studied following stimulation of TLR7 and TLR9 using agonists (imiquimod and CpG-ODN respectively) and inhibition with a specific antagonist (IRS-954) or chloroquine. The effect of these interventions was confirmed in a xenograft model and diethylnitrosamine (DEN)/nitrosomorpholine (NMOR)-induced model of HCC. TLR7 and TLR9 expression was up-regulated in human HCC tissue. Proliferation of HuH7 cells in vitro increased significantly in response to stimulation of TLR7. TLR7 and TLR9 inhibition using IRS-954 or chloroquine significantly reduced HuH7 cell proliferation in vitro and inhibited tumour growth in the mouse xenograft model. HCC development in the DEN/NMOR rat model was also significantly inhibited by chloroquine (P<0.001). The data suggest that inhibiting TLR7 and TLR9 with IRS-954 or chloroquine could potentially be used as a novel therapeutic approach for preventing HCC development and/or progression in susceptible patients.

P92 NEWLY ESTABLISHED MURINE HEPATIC ANGIOSARCOMA CELL LINES: IN VITRO AND IN VIVO MODEL TO STUDY ANGIOSARCOMA BIOLOGY AND TARGETED THERAPIES

was to determine whether TLR7 and TLR9 are potential targets for prevention and progression of HCC. Methods: Tissue microarrays containing liver samples from patients with cirrhosis, viral hepatitis and HCC were examined for expression of TLR7 and TLR9 and the data was validated in liver specimens from the hospital archives. Proliferation of human HCC cell lines was studied following stimulation of TLR7 and TLR9 using agonists (imiquimod and CpG-ODN, respectively) and inhibition with a specific antagonist (IRS-954) or chloroquine. The effect of these interventions was confirmed in a xenograft model and diethylnitrosamine (DEN)/nitrosomorpholine (NMOR)-induced model of HCC. Results: TLR7 and TLR9 expression was up-regulated in human HCC tissue. Proliferation of HuH7 cells in vitro increased significantly in response to stimulation of TLR7. TLR7 and TLR9 inhibition using IRS954 or chloroquine significantly reduced HuH7 cell proliferation in vitro and inhibited tumour growth in the mouse xenograft model. HCC development in the DEN/NMOR rat model was also significantly inhibited by chloroquine (P < 0.001). Conclusions: The data suggest that inhibiting TLR7 and TLR9 with IRS-954 or chloroquine could potentially be used as a novel therapeutic approach for preventing HCC development and/or progression in susceptible patients.

English

Nitrites and morpholine are ubiquitous environmental contaminants found in drinking water and food. NMOR can be formed endogenously from nitrite and morpholine. Increased levels of reactive oxygen species/ reactive nitrite species (ROS/RNS) are involved in the mechanism of NMOR toxicity. Certain antimicrobial, antifungal and antioxidant potential were observed in heterocyclic benzimidazole derivatives and dimethyl sulfoxide (DMSO). This study was designed to evaluate the biological potential of 3-aminothiazolo[3-2a]benzimadzole-2-carbonitrile in the protection of lung and colon tissues against the increased levels of ROS/RNS that are induced by administration of nitrite and morpholine in drinking water for 15 weeks. Forty adult male rats were categorized into 4 groups, 10 rats each. The results showed a significant increase in NO, lipid peroxidation (LPO), total peroxides (TPO), superoxide anion (O2-) and DNA fragmentation in lung and colon tissues of rats treated with nitrite and morpholine compared to the control group. Moreover, histological observation of the lung and colon tissues showed cell necrosis, increase in the leukocyte infiltration and blood vessel congestion. Immunostaining for inducible nitric oxide synthase (iNOS) showed positive reaction for lung and colon tissues. After the co-treatment of rats with DEMSO and 3-aminothiazolo[3-2a]benzimadzole-2-carbonitrile, all the previous biochemical changes were reduced in addition to the relative improvement in the morphological changes of both lung and colon. In conclusion, the injury in lung and colon tissues induced by nitrite and morpholine may return to the increased production of ROS and to the alterations in the levels of antioxidants. Co-treatment of rats with 3-aminothiazolo[3-2a]benzimadzole-2-carbonitrile and DMSO may protect them against nitrite and morpholine toxicity. Key words: Nitrite, morpholine, nitrosomorpholine (NMOR), inducible nitric oxide synthase (iNOS), benzimidazole derivatives, dimethyl sulfoxide (DMSO), colon, lung, rat.

DNA lesions and cytogenetic changes induced by N-nitrosomorpholine in HepG2, V79 and VH10 cells: the protective effects of Vitamins A, C and E

Introduction: N -Nitrosomorpholine (NMOR), present in the workplace of tyre chemical factories, is a known hepatocarcinogen. This compound belongs to the group of N -nitrosamines, which are indirect-acting and require metabolic activation. However, the mechanism of its carcinogenic effect is not completely clear. Aims: The objective of this study was (i) to compare the DNA-damaging and clastogenic effects of NMOR in three cell lines (HepG2, V79 and VH10) with different levels of metabolizing enzymes and (ii) to determine the protective effects of Vitamins A, C and E against deleterious effects of NMOR. Methods: The exponentially growing cells were pre-treated with Vitamins A, C and E and treated with NMOR. Genotoxic effects of NMOR were evaluated by single-cell gel electrophoresis (SCGE, comet assay), while the chromosomal aberration assay was used for the study of clastogenic effects. Key results: NMOR-induced a significant dose-dependent increase of DNA damage as analyzed by SCGE, but the extent of DNA migration in the electric field was unequal in the different cell lines. Although the results obtained by SCGE confirmed the genotoxicity of NMOR in all cell lines studied, the number of chromosomal aberrations was significantly increased only in HepG2 and V79 cells, while no changes were observed in VH10 cells. In HepG2 cells pre-treated with Vitamins A, C and E we found a significant decrease of the percentage of tail DNA induced by NMOR. The reduction of the clastogenic effects of NMOR was observed only after pretreatment with Vitamins A and E; Vitamin C did not alter the frequency of NMOR-induced chromosomal aberrations under the experimental conditions of this study. Conclusions: The fat-soluble Vitamins A and E, which are dietary constituents, reduce the harmful effects of N -nitrosomorpholine in human hepatoma cells HepG2, which are endowed with the maximal capacity for metabolic activation of several drugs.

Histological types of laryngotracheal tumors induced in Syrian golden hamsters by nitrosomorpholine and nitrosopiperidine

The chronic oral administration of nitrosomorpholine (NM) and nitrosopiperidine (NP) to 360 adult Syrian golden hamsters of both sexes resulted in the induction of a high incidence of laryngo-tracheal tumors, which in the group treated with 0.01% solution of NM affected up to 80% of the males. A total of 232 different laryngo-tracheal tumors were found. 36 of these were located within the supraglottic and glottic larynx, 62 were within the subglottic and glottic larynx, and 134 arose within the trachea. Among the laryngeal supraglottic and glottic tumors there were 18 squamous cell carcinomas, 16 squamous cell papillomas and only 2 mixed cell mucoepidermoid papillomas. All the subglottic laryngeal tumors and tracheal tumors were mixed cell mucoepidermoid papillomas. In this study, the squamous cell carcinomas observed within the larynx of hamsters bear morphological and biological similarities to their tumor counterpart found in the larynx of adult man. The laryngeal squamous papillomas observed in this experiment are not similar to the squamous papillomas occurring in man during infancy and childhood. The mixed cell mucoepidermoid papillomas of the hamster trachea and subglottis are a peculiar neoplasm of this species, which morphologically and biologically differs from the cases of tracheal papillomatosis described in man, as well as from the mucoepidermoid tumors of major and minor salivary gland origin also seen in this latter species.

Inhibitory effect of alpha-tocopherol on the formation of nitrosomorpholine in mice treated with morpholine and exposed to nitrogen dioxide.

The possibility that alpha-tocopherol (vitamin E) inhibits the formation of nitrosomorpholine (NMOR) in vivo was investigated in mice orally pretreated with alpha-tocopherol (2.5-100 mg/kg body wt) once daily for 6 days. Twenty-four hours later, the animals were injected i.p. with 2 mg of morpholine (MOR) per animal followed by exposure to 47 p.p.m. of NO2 for 2 h. Under these conditions, low oral doses of alpha-tocopherol (2.5-5 mg/kg body wt) significantly decreased NMOR formation in vivo. As total body alpha-tocopherol levels increased, in vivo NMOR formation decreased, and a maximal 50-70% inhibition of NMOR formation occurred at alpha-tocopherol levels of 5 micrograms/g body wt. Additional results showed that NMOR was rapidly eliminated in mice, so that studies which measure the levels of NMOR found in animals treated with MOR and then exposed to NO2 may underestimate the amount of NMOR that is actually formed. Finally, oral pretreatment of up to 100 mg of alpha-tocopherol/kg body wt had no effect on NMOR elimination.

Nitrosamine metabolism in kwashiorkor rats

The in vitro metabolism of nitrosodimethylamine (NDMA) was studied in liver tissue obtained from male weanling kwashiorkor wistar rats. The elimination of this compound and that of nitrosomorpholine (NMOR) from the blood, after a single intravenous dose, was also investigated. N-demethylase activity in liver microsomes of the test animals was not significantly different from that of the controls although the activity of this enzyme per gram wet liver tissue was considerably reduced in the model animals. On the other hand, the glutathione (GSH) content in liver cytosol of the kwashiorkor animals was much higher than that of the controls. The elimination of NDMA and NMOR from the blood of the experimental animals over 8 hr following i.v. administration of the carcinogens, showed that the clearance rate of each nitrosamine was significantly lower in the kwashiorkor rats.

Carcinogenic effect of nitrosomorpholine administered in the drinking water to Syrian golden hamsters

The long term carcinogenic effect of nitrosomorpholine (NM) was tested in Syrian golden hamsters ( Mesocricetus auratus W.). Groups of 30 females and 30 males were given 0.010%, 0.005% and 0.001% NM in their drinking water. The animals developed neoplasms in the larynx, and trachea (papillary polyps, papillomas and epidermoid carcinomas) and in the liver (hepatocellular adenomas and carcinomas). In addition to these, cholangiocellular and endothelial liver tumours were observed. The overall tumour frequency was dose dependent.

Greater Effectiveness of Hepatocyte and Liver S9 Preparations From Hamsters Than Rat Preparations in Activating N-Nitroso Compounds to Metabolites Mutagenic to &lt;italic&gt;Salmonella&lt;/italic&gt;&lt;xref ref-type="fn" rid="FN2"&gt;2&lt;/xref&gt;&lt;xref ref-type="fn" rid="FN3"&gt;3&lt;/xref&gt;

A comparison was made of the ability of liver S9 and hepatocyte preparations from noninbred Syrian golden hamsters and noninbred Sprague-Dawley rats to metabolically activate a number of nitroso compounds in the Salmonella mutagenesis assay. The liver S9 and hepatocyte preparations from hamsters were consistently more effective than were preparations from rats in metabolizing nitrosodimethylamine (NDM), nitrosodiethylamine, nitrosodiallylamine, nitrosopyrrolidine (NP), nitrosomorpholine (NM), nitrosodiethylmethylurea (NDEMU), and nitrosodimethyl-ethylurea (NDMEU) to mutagenic forms. The use of hamster S9 preparations with NP and NM resulted in up to 14 times the number of revertant colonies obtained with rat preparations; in the presence of hamster hepatocytes, up to 32 times the number of revertants were obtained. The S9 preparations from male hamsters not treated with the enzyme inducers phenobarbital and Aroclor 1254 were more effective than were those from female hamsters for activating NP, NM, and NDM, NDEMU and NDMEU, which have been reported to be carcinogens but not mutagens, were mutagenic in the presence of induced liver S9 or hepatocyte preparations from hamsters but not from rats. When tested with any of the S9 or hepatocyte preparations, nitrosodiphenylamine and nitrosomethylaniline, also reported to be carcinogens but not mutagens, gave no mutagenic responses. Nitrosodioctyl-amine, which has been reported to be noncarcinogenic, was also not mutagenic.

Quantitative determination of half-lifetimes and mutagenic concentrations of chemical carcinogens using a diffusion bioassay

It is possible to deduce quantitative information about the mutagenic concentrations of direct carcinogens and procarcinogens from the well-known experiment in which a droplet of a chemical is put at the center of a petri dish containing a bacterial lawn in an agar gel. The model employed relates the mutagenic action to the space-time concentration profile of the diffusing substances. The range of mutagenic concentrations, in particular the lower limit C mut, at which mutations occur, can be calculated from knowledge of the radii of the mutagenic zone. The only parameters necessary for the calculation of C mut are the diffusion coefficient, D, which can be calculated and the half-life-time of the mutagenic substance, τ 1 2 , which can be obtained from the diffusion experiments. The system was tested with N-methyl- N'-nitro- N-nitrosoguanidine (NG), N-methyl- N-nitrosourea (MNU), ethyl methanesulfonate (EMS), and acetoxy-dimetylnitrosoamine (AcDMN), which are direct carcinogens, and with nitrosomorpholine (NM) and nitrosopyrrolidine (NP), which are procarcinogens. As a consistency check, homogenous experiments (dose-response curves) at low concentrations of the chemicals were also conducted. However, the statistical analysis of the results showed that the homogenous assay is not adequate for determining threshold concentrations. Using the concept of the threshold to rank the tested chemicals according to mutagenic potency, the ranking is: NG > NP > MNU > NM > AcDMN > EMS.

Induction of mouse lung adenomas by amines or ureas plus nitrite and by N-nitroso compounds: effect of ascorbate, gallic acid, thiocyanate, and caffeine.

Lung adenomas were induced in strain A mice by chronic treatment with N-nitroso compounds (given in drinking water) and with amines or ureas in food plus NaNO2 in drinking water. We studied the effects of varying the concentrations of three N-nitroso compounds and NaNO2 concentration in the morpholine plus NaNO2 and methylurea plus NaNO2 systems. Sodium ascorbate (NaASC) at the highest level tested (11.5 or 23 g/kg food) gave 89-98% inhibition of adenoma induction by the NaNO2 plus piperazine, morpholine, and methylurea systems. In 7 groups, NaASC produced increases of 15-59% in adenoma induction by nitrosomorpholine (NM) and mononitrosopiperazine (MNP), possibly because the mice consumed more of the nitrosamine solution. Adenoma induction by morpholine plus NaNO2 was strongly inhibited by gallic acid, moderately inhibited by caffeine, and unaffected by thiocyanate (all added to the food). Gallic acid inhibited or had no effect on the action of NM and MNP. We discussed the proposal that NaASC (or perhaps gallic acid) be administered with readily nitrosatable drugs.

Repair of lesions induced in human liver cell DNA by N-nitroso compounds as measured by the bromodeoxyuridine photolysis method

Chang human liver cells were treated with the carcinogens N-methyl- N′-nitrosoguanidine (MNNG) and nitrosomorpholine (NM). In addition, cells were exposed to the folic acid analog, 2-hydroxy- N 10 nitrosofolic acid. Repair of the damage to DNA was estimated by selective photolysis of BUdR-containing repaired regions with 313 nm radiation. The influence of the co-carcinogen Arlacel A was estimated with the three compounds. Results indicated significant repair synthesis with MNNG- and NM-treated cells. 2-Hydroxy- N 10 nitrosofolic acid elicited no damage to the liver DNA. Arlacel A prevented repair synthesis in cells treated with NM and MNNG.

Carcinogenic effects of N-nitrosomorpholine and N-nitrosopiperidine on European hamster (Cricetus cricetus).

The effects of nitrosomorpholine (NM) and nitrosopiperidine (NP) on 60 male and 60 female captured European hamsters (Cricetus cricetus) were investigated. After the mean lethal dose (LD50) wasdetermined for each substance, animals were treated with varying doses of the LD50 of either NM or NP. Both NM and NP proved to be markedly effective carcinogens as they caused a high incidence of multiple primary neoplasms in more than one organ. These neoplasms developed most frequently in the respiratory and upper digestive tracts. Moreover, a high percentage of the tumors in the nasal and oral cavities, as well as the lungs, underwent malignant transformations. NP application also induced a high frequency of squamous cell carcinomas originating from the epithelium of the gingiva propria.

Repair of lesions induced in human liver cell DNA by N-nitroso compounds as measured by the bromodeoxyuridine photolysis method

Chang human liver cells were treated with the carcinogens N -methyl- N ′-nitrosoguanidine (MNNG) and nitrosomorpholine (NM). In addition, cells were exposed to the folic acid analog, 2-hydroxy- N 10 nitrosofolic acid. Repair of the damage to DNA was estimated by selective photolysis of BUdR-containing repaired regions with 313 nm radiation. The influence of the co-carcinogen Arlacel A was estimated with the three compounds. Results indicated significant repair synthesis with MNNG- and NM-treated cells. 2-Hydroxy- N 10 nitrosofolic acid elicited no damage to the liver DNA. Arlacel A prevented repair synthesis in cells treated with NM and MNNG.