N-nitrosamine formation by macrophages

Nitrate biosynthesis is a known mammalian process, and macrophages from mice treated with Escherichia coli lipopolysaccharide (LPS) have been shown to be capable of nitrate synthesis. Cell culture studies showed that macrophages produce nitrite as well as nitrate. We report here N-nitrosamine formation by stimulated macrophages. Experiments were carried out with the macrophage cell lines, J774.1, WEHI-3 and RAW 264. Macrophages were cultured in Dulbecco's modified Eagle's medium (pH 7.5) supplemented with calf serum (10%). The concentration of nitrate in the supernatant was measured. N-nitrosamines were extracted with dichloromethane and the extracts were analysed by gas chromatography-thermal energy analysis. When J774.1 (1.5 X 10(6) cells/ml) were incubated with LPS (10 micrograms/ml) and morpholine (15 mM) for 72 h at 37 degrees C, N-nitrosomorpholine (NMOR) was produced (0.8 microM). The amount of nitrite produced was 50 microM. RAW 264 and WEHI-3 also produced NMOR; LPS was required for nitrite and NMOR formation. gamma-Interferon (IFN) promoted both NMOR (2.5 microM) and nitrite (70 microM) formation. Nitrite (150 microM) incubated with morpholine and the medium did not form NMOR. Kinetics of LPS-induced nitrite and NMOR formation in J774.1 showed that the rate of NMOR formation was highest in the middle incubation period (24-36 h), although the nitrite concentration was highest in the latter incubation period (48-60 h). Our results showed that macrophages may be capable of nitrosamine formation under physiological conditions that do not normally permit this reaction.     

Endogenous incorporation of nitric oxide from L-arginine into N-nitrosomorpholine stimulated by Escherichia coli lipopolysaccharide in the rat.

The endogenous formation of nitrate in the rat, mouse and human occurs through cellular processes involving the oxidation of the guanido group of arginine. These processes proceed from arginine to nitric oxide with subsequent conversion to electrophilic nitrosating agents capable of forming carcinogenic nitrosamines. We have now demonstrated that endogenous nitrosamine formation can occur via cells stimulated in vivo by bacterial lipopolysaccharide (LPS). The nitrosation of morpholine given to rats by i.p. injection yields N-nitrosomorpholine (NMOR) which is subsequently oxidized in the liver. A major metabolite of NMOR, N-nitroso-(2-hydroxyethyl)glycine, was previously shown by other investigators to be excreted into urine. Treatment of rats with LPS, arginine and morpholine creates a large increase in NMOR urinary metabolites over a 24-h period. This process is not influenced by simultaneous dosage with large amounts of NaNO3. Therefore the endogenous LPS-induced formation of NMOR proceeds directly from nitric oxide prior to incorporation into the nitrate body pool. The proportion of endogenously synthesized nitric oxide incorporated into NMOR is approximately 3 x 10(-6).     

Macrophages produce nitrite, nitrate and nitrosamines after addition of catalase.

Mouse macrophages produced nitrite and N-nitrosomorpholine after incubation with catalase. A macrophage cell line, J774.1 (1 x 10(6) cells/ml), was incubated with catalase (500 U/ml) and morpholine (5 mM); after 48 h incubation at 37 degrees C, macrophages produced nitrite (100 microM) and N-nitrosomorpholine (1 microM). Stimulation of J774.1 cells with catalase enhanced interleukin-1 production and tumour-killing activity against mastocytoma P815 cells. Flow cytometric analysis showed that catalase was bound to the surface of the macrophages.     

Bacterial formation of N-nitroso compounds in the rat stomach after omeprazole-induced achlorhydria

N-Nitrosamine formation by bacteria in the achlorhydric stomach has been proposed as an important factor in the development of gastric cancer. Thus, the effect of the presence of bacteria in the stomach on endogenous nitrosation was investigated in rats given omeprazole (an inhibitor of gastric H+, K((+)-ATPase) which reduces gastric secretion sufficiently to allow survival of a bacterial suspension of Escherichia coli or Pseudomonas. When rats were given both thiazolidine 4-carboxylic acid and nitrate, greater endogenous nitrosamine formation was observed in rats receiving omeprazole and an E. coli suspension than in control or omeprazole-treated rats. A similar result was obtained when rats were given morpholine and nitrate. Since the endogenous formation of N-nitrosomorpholine (NMOR) can be evaluated more precisely from the levels of its urinary metabolites, N-nitrosohydroxyethylglycine (NHEG), the metabolism of NMOR was studied in omeprazole-treated rats. In this preliminary study, we showed that 60% of an oral dose of NMOR was excreted as NHEG, while in rats with a higher gastric pH 20% was excreted as NHEG. The amount of endogenously formed NMOR was increased in omeprazole-treated rats given morpholine and nitrite together with bacteria, and greater excretion of unchanged urinary NMOR was observed. Thus, as shown in this in-vivo model, bacteria efficiently reduce nitrate to nitrite and catalyse nitrosation, resulting in increased endogenous formation of N-nitroso compounds in the achlorhydric stomach.     

In-vivo nitrosation of amines in mice by inhaled nitrogen dioxide and inhibition of biosynthesis of N-nitrosamines

Inhalation of nitrogen dioxide (NO2) by mice administered orally morpholine (MOR) or dimethylamine (DMA) resulted in the biosynthesis of N-nitrosomorpholine (NMOR) or N-nitrosodimethylamine (NDMA), respectively, as determined by the analysis of frozen whole-mouse powder, using gas chromatography with a Thermal Energy Analyzer detector. Significant levels of NMOR were detected following exposure of mice to 0.38 mg/m3 NO2 for 0.5 h (26 ng NMOR/mouse) and there was a two-fold increase when NO2 exposure was extended to 4 h. NMOR levels also increased in a time-dependent manner at 28.4 and 47.3 mg/m3 NO2 exposure levels, reaching a maximum of 450 and 725 ng NMOR/mouse, respectively, at 4 h. Oral administration of sodium ascorbate (50-250 mg), ammonium sulfamate (50-100 mg) or DL-alpha-tocopherol (67-167 mg) immediately after MOR or DMA, but prior to NO2 exposure, significantly inhibited both NMOR and NDMA biosynthesis, sulfamate being the most effective (greater than 90% NMOR and NDMA inhibition), followed by ascorbate (83-90% NMOR and 58-90% NDMA inhibition) and alpha-tocopherol (22-42% NMOR and 46-69% NDMA inhibition). Low levels of NDMA were found in untreated control mice (less than 13 ng/mouse) and in most samples of commercially obtained animal feed (10-15 micrograms/kg); NMOR, however, was not detectable or was detected in negligible amounts in these cases. Various control experiments indicated that most of the recovered nitrosamine resulted from in-vivo nitrosation in mice, with only up to 1-2% of NMOR and approximately 10% of NDMA yields being attributed to artefact formation, possibly during work-up of the mouse-powder samples.     

L-arginine-dependent formation of N-nitrosamines by the cytosol of macrophages activated with lipopolysaccharide and interferon-gamma.

The cytosol fraction of J774-1 murine macrophages activated with lipopolysaccharide (LPS) + interferon-gamma (IFN-gamma) was found to nitrosate a wide range of secondary and tertiary amines. The reaction was dependent on L-arginine and NADPH. The optimal pH for nitrosation was 7.2-7.3. Nitrosation was inhibited by arginine derivatives such as NG-monomethyl-L-arginine and NG-nitro-L-arginine, well-known inhibitors of nitric oxide (NO) synthase. These results indicate that nitrosation is mediated by NO synthase, which catalyzes formation of NO and L-citrulline from L-arginine. Nitrosamine formation also required oxygen and was inversely correlated with the basicity of nitrosatable amines. The nitrosation was inhibited by oxyhemoglobin, an NO trapping agent, and enhanced by superoxide dismutase, which stabilizes NO. LPS + IFN-gamma induced approximately 500-600 times greater nitrosation activity than that of non-activated macrophages. Macrophages treated with LPS alone exhibited 3-4 times greater nitrosation activity than untreated macrophages, whereas macrophages treated with IFN-gamma alone did not show enhanced nitrosation activity. A combination of the cytosols from macrophages treated with LPS alone and IFN-gamma alone did not nitrosate morpholine as rapidly as the cytosol of macrophages treated with both compounds together. The activity for forming L-citrulline and nitrite/nitrate from L-arginine was markedly induced by treatment with either LPS alone or LPS + IFN-gamma but not with IFN-gamma. Those results suggest that some other factor(s) in addition to NO synthase is involved for efficient nitrosation by the macrophage cytosol. This factor(s) was not induced in macrophages by either LPS- or IFN-gamma alone, but was induced only in the presence of the two compounds.     

Nitrate as a precursor of the in-vivo formation of N-nitrosomorpholine in the stomach of guinea-pigs

Reduction of nitrates to nitrites and formation of the carcinogen N-nitrosomorpholine (NMOR) was investigated in the stomach of guinea-pigs. A semisynthetic diet with nitrate plus morpholine was administered intragastrically after a 24-h fast; after treatment, the animals were killed and stomach nitrite contents were determined 6, 12, 18, 24 and 30 min after the treatment using a colorimetric method. NMOR content was determined 18 min after treatment with nitrate plus morpholine using gas chromatography-thermal energy analysis. Reduction of nitrates to nitrites in the stomach was observed that was sufficient to synthesize NMOR in guinea-pigs under the conditions of this experiment.     

In vivo formation of N-nitrosomorpholine in CD-1 mice exposed by inhalation to nitrogen dioxide and by gavage to morpholine

Male CD-1 mice were exposed to approximately 20 ppm nitrogen dioxide (NO2) for 5-6 hours, to 1 g morpholine/kg body weight by gavage, or to both. Treatments were repeated daily for 5 consecutive days. N-nitrosomorpholine (NMOR) was found in whole carcasses (16-146 ng NMOR/mouse) in all animals that had been exposed to both NO2 and to morpholine, but NMOR was not found in tissues from animals that had been exposed to either chemical alone. Approximately one-third of the NMOR was found in the gastrointestinal tract, mainly in the stomach. The coadministration of 2 g sodium ascorbate/kg body weight or 1 g alpha-tocopheryl acetate/kg body weight had no effect on the amount of NMOR that was found in any tissue. Another possible product of the interaction of NO2 and morpholine, N-nitromorpholine, was not detected in any tissue. We concluded that the repeated, concurrent exposures of mice to NO2 by inhalation and to morpholine by gavage resulted in the in vivo formation of significant quantities of NMOR. The biological significance of the observation remains unknown.     

Liver and forestomach tumors and other forestomach lesions in rats treated with morpholine and sodium nitrite, with and without sodium ascorbate

Administration to rats of ascorbate with morpholine and nitrite was previously shown to inhibit the liver tumor production and to enhance the induction of forestomach tumors, as compared to treatment with morpholine and nitrite. In a repetition of this experiment, 10 g morpholine/kg in the diet and 2 g sodium nitrite/liter in the drinking water were administered for life to male MRC-Wistar rats without (group 1) or with (group 2) 22.7 g sodium ascorbate/kg in the diet. Group 3 was untreated. Group 2 showed a lower liver tumor incidence with a longer latency than group 1, indicating a 78% inhibition by ascorbate of in vivo N-nitrosomorpholine (NMOR) formation. The incidence of forestomach papillomas was 3% in group 1, 38% in group 2, and 8% in group 3. The difference between groups 1 and 2 was not significant due to the shorter life-span of group 1. Group 1 and especially group 2 had more forestomach hyperplasia and hyperkeratosis than group 3. Ascorbate might have enhanced induction of these lesions because of an action synergistic with that of NMOR. However, it is most likely that the lowered NMOR dose and concomitantly increased survival produced by the ascorbate were solely responsible for the increased incidence of forestomach papillomas and other lesions in group 2.     

N-nitrosamine formation by microorganisms isolated from human gastric juice and urine: biochemical studies on bacteria-catalysed nitrosation

Twelve out of 14 bacterial strains isolated from patients with urinary infections and nine out of 30 microorganisms isolated from gastric juice from patients with gastric achlorhydria were shown to catalyse the formation of N-nitrosomorpholine (NMOR) from nitrite and morpholine at neutral pH. The effects of various metal ions and cofactors on the bacterial nitrosation reaction was investigated. The presence of nitrate in the culture medium was required to induce nitrosating activity in bacteria, but low nitrate concentrations inhibited the nitrosation reaction.     

Nitrosamine formation from amines applied to the skin of mice after and before exposure to nitrogen dioxide

Skin lipids of mice exposed to NO2 contain lipid-soluble nitrosating agent(s) (NSA) that react in vitro with amines to produce nitrosamines. To test whether this reaction occurs in skin, we exposed mice to 50 ppm NO2 for 4 h and, 20 h later, applied 25 mg morpholine or N-methylaniline to the skin, which was then analyzed for the corresponding nitrosamine. When morpholine was applied, mean N-nitrosomorpholine yield was only 0.3 nmol/mouse (not significant). When N-methylaniline was applied and mice were killed after 10-40 min, N-nitroso-N-methylaniline yield in the skin was 13-21 nmol/mouse of which 87% occurred in the hair. NSA formation when mice were exposed to 6.5 ppm NO2 was only 0.15% of that for exposure to 50 ppm NO2. NSA occurred mostly in surface lipids of the skin and its in vitro reaction to give nitrosamines was not inhibited by alpha-tocopherol. When morpholine was painted and mice were then exposed to 55 ppm NO2 for 30 min, the skins contained 19 nmol N-nitrosomorpholine/mouse, attributed to a direct reaction between NO2 and the amine. We concluded that nitrosamine formation in skin by this direct reaction may be more important than the reaction of amines with NO2-derived NSA.     

Further studies on murine macrophage synthesis of nitrite and nitrate

Further studies on macrophage synthesis of nitrite and nitrate showed lipopolysaccharide (LPS) and interferon (IFN) to be potent stimuli. Kinetic experiments showed a time lag of 6 h for LPS and 10-12 h for IFN. The protein synthesis inhibitor cycloheximide completely inhibited nitrite and nitrate synthesis when present in the media at time 0 but had no effect if added at times after the lag period. A number of experiments were carried out to test the involvement of reactive oxygen species (the 'oxygen burst') in stimulated macrophage synthesis. All results were consistent with a lack of involvement of the oxygen burst in this process.     

Synthesis of nitric oxide and nitrosamine by immortalized woodchuck hepatocytes

Woodchuck (Marmota monax) hepatic cells, which were immortalizedby the simian virus 40 large T antigen (SV40 Tag) produced nitricoxide (NO; measured as nitrite) in vitro from L-arginine (L-Arg)after lipopolysaccharide (LPS) treatment. NO synthesis was relatedto L-Arg and LPS concentration and plateaued at 1.0 mM L-Argand 1.0 µg/ml LPS. LPS-stimulated cells nitrosated morpholineto form N-nitrosomorpholine (NMOR) in the presence of L-Argat pH 7.4. NMOR production increased 7-fold in LPS stimulatedcells compared to unstimulated hepatocytes. N-nitroso-dimethylamine(NDMA) was detected in the cell culture medium in the presenceof LPS and L-Arg but without added dimethylamine. N^G-monomethyl-L-arginine,a selective inhibitor of nitric oxide synthase, inhibited formationof NO and NMOR, indicating that NO and nitrosating agents wereformed via the L-Arg-nitric oxide pathway. These data are thefirst to report NO and N-nitrosamine production by immortalizedhepatocytes and confirm earlier work showing that primary hepatocytesform NO in culture. This suggests that hepatic formation ofN-nitroso compounds and/or NO could be an etiologic factor inhepatocellular carcinoma. Immortalized woodchuck hepatic cellsmay be useful as in vitro models to study the L-Arg-nitric oxidepathway and its possible role in liver carcinogenesis.     

Synthesis of nitrite and nitrate in murine macrophage cell lines

Synthesis of nitrite (NO2-) and nitrate (NO3-) was studied in the macrophage cell lines RAW 264.7, WEHI-3, PU5-1.8, J774A.1, and P388D1 and compared to the synthesis by thioglycolate-elicited peritoneal macrophages from C3H/He and C3H/HeJ mice. Treatment with Escherichia coli lipopolysaccharide (LPS) induced NO2-/NO3- synthesis by all the cell lines except P388D1, which remained unresponsive at the highest LPS concentration (50 micrograms/ml). Recombinant murine gamma-interferon induced NO2-/NO3- synthesis in only two cell lines (PU5-1.8 and RAW 264.7), although it activated synthesis by C3H/He and C3H/HeJ macrophages. Dual signal treatments consisting of lymphokines or gamma-interferon plus LPS stimulated NO2-/NO3- synthesis by all five cell lines and each line showed enhanced synthesis as compared to that induced by any single stimulus. Heat-killed Bacillus Calmette-Guérin and purified mycobacterial protein derivative stimulated NO2-/NO3- synthesis in three of five cell lines, while dextran sulfate, zymosan, and the synthetic adjuvant muramyl dipeptide were ineffective. Nitrite represented 50-75% of the total NO2-/NO3- produced in all cases. The kinetics of LPS-induced NO2-/NO3- synthesis in J774A.1 and C3H/He macrophages were identical; a 6-h lag phase was followed by a 24- to 48-h period in which NO2- and NO3- were in a ratio of approximately 3:2 at all time points.     

N-nitrosation and N-nitration of morpholine by nitrogen dioxide: inhibition by ascorbate, glutathione and alpha-tocopherol

Ascorbate anion and glutathione were found to inhibit the aqueous reaction between nitrogen dioxide (NO2) and morpholine (MOR) and thereby prevented the formation of N-nitrosomorpholine (NMOR) and N-nitromorpholine (NTMOR) at both pH 7.4 and 12.5. These antioxidants are approximately 3 orders of magnitude more reactive towards NO2 than is MOR and may play an important role in the prevention of carcinogen formation in the lung due to inhaled NO2. Ammonium sulfamate was ineffective at preventing nitrosation or nitration by NO2 at either pH 7.4 or 12.5.     

A nitrosating agent from the reaction of atmospheric nitrogen dioxide (NO2) with methyl linoleate: comparison with a product from the skins of NO2-exposed mice

We showed previously that exposure of mice to atmospheric nitrogen dioxide (NO2) leads to the formation of an ether-extractable nitrosating agent (NSA) in the skin, which produced N-nitrosomorpholine (NMOR) from morpholine in vitro but not in vivo (under our conditions). We now report that NO2 bubbled into hexane solutions of methyl linoleate (MLIN) produced a similar NSA that reacted with morpholine in dichloromethane solution to produce NMOR. The NSA yield increased sharply as MLIN concentration was raised, with a maximum 0.1% yield of NSA from MLIN. The NSA yield from MLIN was four times that from methyl oleate and seven times that from methyl stearate. The NSA derived from MLIN travelled on thin-layer chromatography more slowly than the main weight fraction; whereas TLC of NSA in the skin lipids of NO2-exposed mice and in untreated mouse skin lipids exposed in vitro to NO2 produced NSA that travelled more rapidly than the main weight fraction.     

Effects of phenol and 2,6-dimethoxyphenol (syringol) on in vivo formation of N-nitrosomorpholine in rats

We determined the effects of phenol and 2, 6-dimethoxyphenol(syringol) on N-nitrosomorpholine (NMOR) formation in rats givenmorpholine and nitrite by gavage. At 30 min post-gavage therecovery (from the stomach, duodenum and blood) of 564 µgNMOR was six times higher when administered to rats by gavagewith 2 g of semipurified diet (SPD) than when given withoutfood. Rats were gavaged with 12 mg each of morpholine, one ofthe modifiers and nitrite and examined 30 min later. Syringoldecreased the amount of NMOR in both the stomach and blood by89%, while phenol had no effect. We compared these results withthose obtained with ascorbic acid and thiocyanate. The effectof ascorbic acid was similar to that of syringol. However, thiocyanateincreased the amount of NMOR in the stomach and blood 2.7- and4-fold, respectively. When 2 g of SPD was administered to ratsby gavage, together with the precursors, syringol and ascorbicacid blocked NMOR formation in the stomach by 58 and 45%, respectively,and thiocyanate enhanced the yield 1.5-fold. The effect of phenolwas not significant for the stomach and blood and that of theother modifiers was not significant for blood. Administrationof the reactants together with food decreased the NMOR levelin blood 155-fold relative to controls (no food), suggestingthat food decreased the absorption rate over a 30-min period.These results demonstrate the modifying effect of phenol andsyringol on NMOR formation in vivo to be similar to that observedin a previous in vitro study, and show that the effect of foodon NMOR levels in blood was more important than that of themodifiers.     

Experimental model for evaluating animal exposure to endogenous N-nitrosodi-n-butylamine by measuring its urinary metabolites N-butyl-N-(4-hydroxybutyl)-nitrosamine and N-butyl-N-(3-carboxypropyl)nitrosamine

Endogenous formation of N-nitrosodi-n-butylamine (NDBA) was studied in rats after administration of sodium nitrite or sodium nitrate and N,N-dibutylamine (DBA) by monitoring the urinary excretion of NDBA and its metabolites, N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) and N-butyl-N-(3-carboxypropyl)nitrosamine (BCPN). Animals were given sodium nitrite (0.2%) or sodium nitrate (0.5%), dissolved in the drinking-water. This treatment was started 24 h before DBA administration and was continued throughout the experiment. Animals were fasted overnight before receiving DBA, which was administered by gavage as three doses of 50 mg/kg, 8 h apart; 24-h urine samples were collected on ammonium sulfamate. NDBA, BBN and BCPN were extracted and analysed by GC-TEA, according to a method previously described. Under the experimental conditions reported, NDBA and BBN (free or glucuronic acid-conjugated) were not detected in the urine of animals given nitrite or nitrate and DBA, but the presence of BCPN indicated that N-nitrosation had occurred in both groups of animals. These results suggest that, when studying nitrosamines that are extensively metabolized, quantitative analysis of urinary metabolites is a better indicator of nitrosamine exposure than measurement of nitrosamine itself.     

Induction of reproductive system tumors in mice by N6-(methylnitroso)-adenosine and a tumorigenic effect of its combined precursors.

Interaction of the naturally-occurring nucleoside, N6-methyl adenosine, with nitrite, a reaction that occurs readily under acidic conditions, results in the formation of a nitrosamine, N6-(methylnitroso) adenosine[m6(NO)Ado]. This nitrosamine was given in the drinking water (1 mM solution) of non-inbred Swiss mice from 3 weeks of age until death. It caused a significant increase in the incidence of primary lung tumors, compared with controls. It also induced reproductive tract tumors in 80% of the exposed females, including mammary tumors in 60% and uterine tumors in 25%. The precursors of m6(NO) Ado, m6Ado and nitrite, did not elevate tumor incidence when given singly, but when administered together resulted in a significant increase in numbers of lung tumors in the males. The nitrosamine base, N6-(methylnitroso)adenine, was found to be a less potent carcinogen than m6(NO)Ado, causing lung tumors only in males and possibly a few mammary tumors in females. These results indicate the in vivo formation of a carcinogen from m6Ado and nitrite, and show that m6(NO)Ado induces neoplasms in the reproductive system of mice, an unusual target for a N-nitroso carcinogen.