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Alkylating activity in food products--especially sauerkraut and sour fermented dairy products--after incubation with nitrite under quasi-gastric conditions.

N-Nitroso compounds may well rank high among the genotoxic carcinogens present in our environment. Small amounts of such compounds may be formed in the human stomach after consumption of high-nitrate vegetables. Volatile nitrosamines can be conveniently determined but reliable methods of analysis for non-volatile N-nitroso compounds are still lacking. In this study we have used the 4-(4-nitrobenzyl)pyridine test to look for the formation of alkylating compounds such as N-nitroso-N-methylurea in a wide range of food products after incubation with nitrite under simulated gastric conditions. Our results indicate that many food products do not form alkylating compounds in appreciable amounts, even though the nitrite concentration used was five to ten times that found in saliva after a high-nitrate meal. Comparatively strong alkylating activity, however, was detected after incubation of samples of sauerkraut, certain dairy products (yoghurt, biogarde, quark, buttermilk and milk), wine and smoked mackerel. Samples of sauerkraut incubated with simulated gastric fluid, but without (added) nitrite, also displayed appreciable alkylating activity. The formation of alkylating substances in non-fat yoghurt was markedly inhibited by addition of ascorbic acid. The identity of the alkylating agents remains unknown. The isolation procedure was optimized for N-nitroso-N-methylurea, but will certainly result in the isolation of other compounds, such as C-nitroso-, C-nitro- or perhaps even C-nitroso-C'-nitro-compounds as well. Biogenic amines, glucosinolates, indole derivatives or other compounds may be involved as precursors. If alkylating agents are formed in vivo after ingestion of high-nitrate vegetables or drinking water, this is likely to occur only when the food products mentioned above are ingested simultaneously with or shortly after the nitrate load and not appreciably (except perhaps in the case of sauerkraut) when they are ingested alone, without a nitrate source. The health implications of these findings cannot yet be established. Many alkylating agents, however, have strong carcinogenic properties and continued investigation of food products (and their interaction products with nitrite) is indicated.

Alkylating Agents↗

Interactive mutagenicity of sodium nitrite, dimethylamine, methylurea and ethylurea.

Groups of mice were treated per os with sodium nitrite either alone or in combination with nitrosatable amino compounds and tested in the host mediated assay. When mice were treated with sodium nitrite in combination with dimethylamine a small(4-fold) but significant increase in mutant frequency (MF) was observed. Ethylurea or methylurea in combination with sodium nitrite induced 10- or 850-fold increases in MF, respectively. The response to methylurea was dose-dependent with a 6- and 30-fold increase in MF at 5.4 and 11.5 mg/kg NaNO2 and a 6-fold increase at 108 mg/kg methylurea. That this response reflected gastric nitrosation was shown by the disappearance of the response if NaNO2 administration preceded methylurea treatment by 10 min. High MF's were observed if NaNO2 was administered 10 or 20 min after methylurea.

Animals↗

Rapid mineralisation of the herbicide isoproturon in soil from a previously treated Danish agricultural field.

Mineralisation of the phenylurea herbicide isoproturon (3-(4-isopropylphenyl)-1,1-dimethylurea) and two of its known metabolites, 3-(4-isopropylphenyl)-1-methylurea (monodesmethyl-isoproturon) and 4-isopropylaniline, was studied in Danish agricultural soils with or without previous exposure to isoproturon. A potential for rapid mineralisation of isoproturon and the two metabolites was present in soils sampled from three plots within an agricultural field previously treated regularly with the herbicide, with 34-45%, 51-58% and 33-36% of the added [phenyl-U-14C]isoproturon, [phenyl-U-14C]monodesmethyl-isoproturon and [phenyl-U-14C]4-isopropylaniline metabolised to [14C]carbon dioxide within 30 days at 20 degrees C. In contrast, such extensive mineralisation of these three compounds was not observed within this period in soils sampled from two other agricultural fields without previous treatment with isoproturon. The mineralisation patterns indicated growth-linked metabolism of the three compounds in the previously exposed soils, and doubling times for [14C]carbon dioxide production ranged from 1.6 to 3.2, 1.0 to 2.1 and 1.3 to 1.7 days for isoproturon, monodesmethyl-isoproturon and 4-isopropylaniline, respectively. The ability to mineralise [phenyl-U-14C]isoproturon to [14C]carbon dioxide was successfully sub-cultured to a fresh mineral medium which provided isoproturon as sole source of carbon and nitrogen. One of the soils sampled from an agricultural field not previously treated with isoproturon showed accelerated mineralisation of [phenyl-U-14C]4-isopropylaniline toward the end of the experiment, with a doubling time for [14C]carbon dioxide production of 7.4days. This study indicates that the occurrence of rapid mineralisation of the phenyl ring of isoproturon to carbon dioxide is related to previous exposure to the herbicide, which suggests that microbial adaptation upon repeated isoproturon use may occur within agricultural fields.

Agriculture↗

A comparative study on the effect of MNU on human lymphocyte cultures in vitro evaluated by O6-mdG formation, micronuclei and sister chromatid exchanges induction.

N-Nitroso-compounds are a large group of chemicals present in a number of environmental sources and many of them are mutagens as well as carcinogens in experimental animals. Among the known N-nitroso-compounds, N-nitroso-N-methylurea (MNU) is a strong mutagen. In this study an effort has been made to compare the ability of MNU to methylate the O6-guanosine site in DNA and to induce micronuclei and sister chromatid exchanges in human lymphocyte cultures in vitro. To quantitate O6-methyldeoxyguanosine (O6-mdG) a highly sensitive immunoassay, immuno-slot-blot (ISB), has been used. For the evaluation of micro nuclei (MN) the cytokinesis block micronucleus method has been used. Different concentrations (75, 100, 125 micrograms/ml) were tested. At the highest concentration tested for the MN induction, 125 micrograms/ml, the occurrence of binucleates and micro nuclei is higher than twice in relation to control and a reduction in NDI is also observed. The same concentrations were used for the estimation of sister chromatid exchanges (SCEs) induction. The mean number of SCEs at 125 micrograms/ml is almost three times that of the control level. The concentrations tested for the quantitation of O6-mdG were 200, 300 and 400 micrograms/ml and this was done because for the test system we used and for the given experimental conditions the first indication of O6-mdG formation was at 200 micrograms/ml. Our results show that methylation of O6-guanosine increases with concentration and at 400 micrograms/ml the concentration of O6-mdG is 5.83 fmol/microgram DNA, while at the control level it is 2.40 fmol/microgram DNA. Since O6-mdG formation is observed in higher concentrations than those of MN and SCE induction it would be interpreted that O6-mdG levels are not correlated with the studied cytogenetic effects although one has to take into consideration the total promutagenic lesions in DNA, induced by MNU, as well as AGT repair activity.

Carcinogens↗

[The mechanisms of neutrophilic oxygen damage in ischemic and reperfusion lesions. Pharmacological manipulations].

This work studies the ischemic/reperfusion injury (I/R) in relationship to the production of free radicals from activated neutrophils. It is well known, that biochemical and physiopathological alterations participate in the tissue lesion produced by the interaction oxygen/leukocyte. Pharmacological manipulations, oriented to eliminate the harmful effect of the free radicals, remain as the most significant event in the management of the I/R injury. Compounds such as allopurinol, adenosine, naloxone, corticosteroids, methylurea, and others, have demonstrated a protective effect during the I/R injury. Although, the mechanism of action of these compounds is unknown, it is possible that the free radical scavenging effect, could be the most important protective mechanism. The better understanding of the I/R injury would allow for a better protection of the damaged organ.

Animals↗

[The role of bacterial metabolism in transformation of non-mutagenic compounds into mutagens. I. Participation of Escherichia coli nitroreductase in creation of mutagens from non-mutagenic new pesticides].

Investigations concerned Escherichia coli nitroreductase in creation of mutagens from non-mutagenic pesticides-derivatives of urea. Three new compounds were studied: N-phenyl-N'-methylurea (IPO 4328), N-methyl,N-(2-hydroxyethyl)-N'phenylurea (IPO 2363), N-(2-hydroxyethyl), N-methyl-N'-(3,4 dichloroethyl) urea, and diurone-3-(3,4 dichlorophenyl)-1,1 dimethylurea. These compounds were incubated in anaerobic conditions with cells of E. coli K-12 (KF) strain and nitrate or nitrite. Using Ames test, mutagenicity of resulting metabolites was investigated. It was found that during incubation of herbicide IPO 4328 with cells of E. coli K-12 (KF) and nitrate, mutagenic product for strain of S. typhimurium TA 1537 is created. Very weak mutagenic metabolite for the same strain was appearing during incubation of herbicide IPO 2363 with cells of E. coli K-12 (KF) in presence of nitrite. Incubation of investigated compounds with E. coli K-12 (KF) cells alone did not result in appearance of mutagenic substances. Thus, role of Escherichia coli in creation of mutagenic compounds from non-mutagenic derivatives of urea consisted of nitrite from nitrate production with participation of nitroreductase, which afterwards in absence of bacteria or action of their enzymes reacted with investigated pesticides.

Escherichia coli↗

Incidence of p53 and Ha-ras gene mutations in chemically induced rat mammary carcinomas.

To determine whether p53 alterations, which are frequent in human breast cancers, are also common in rat mammary tumors, we examined 40 tumors from 24 rats treated with 7,12-dimethylbenz[a]anthracene (DMBA) and 34 tumors from 14 rats treated with N-nitroso-N-methylurea (NMU) (an N-nitroso compound). DMBA and NMU are known genotoxic mutagens. The entire coding regions of the p53 and Ha-ras genes were examined for mutations by polymerase chain reaction single-strand conformational polymorphism analysis and by direct sequencing. One of the 40 DMBA-induced mammary tumors had a p53 mutation, a single-base substitution (AGC-->GGC) at codon 307, resulting in an amino-acid change from Ser to Gly. No mutations were found in NMU-induced tumors. The incidence of Ha-ras gene mutation was 79% (27 of 34) at codon 12 in the NMU group and 23% (nine of 40) at codon 61 in the DMBA group. Thus, p53 mutation, in contrast to Ha-ras mutation, did not seem to be a prerequisite for carcinogenesis in chemically induced rat mammary tumors.

9,10-Dimethyl-1,2-benzanthracene↗

Chromosomal aberrations and sister-chromatid exchanges induced by N-nitroso-2-acetylaminofluorene and their modifications by arsenite and selenite in Chinese hamster ovary cells.

The frequencies of chromosomal aberrations (CA) and sister-chromatid exchanges (SCE) in Chinese hamster cells were significantly increased by the direct-acting mutagen N-nitroso-2-acetylaminofluorene (N-NO-AAF) at the concentration of 0.1 mM. N-NO-AAF was prepared by nitrosation of the protohepatocarcinogen 2-acetylaminofluorene. The induced CA, which included chromatid breaks, chromatid exchanges, chromosome breaks, and chromosome ring formation were significantly potentiated by the presence of sodium arsenite (10 microM), but not by hydroxyurea (20 mM) or cytosine arabinoside (25 microM). On the other hand, the clastogenic effect of N-NO-AAF was effectively inhibited by sodium selenite (100 microM). Arsenite (10 microM) was shown to be moderately active in CA induction which was partially blocked by the presence of selenite (10 nM). N-Nitroso compounds such as N-nitroso-N-methylurea, N-nitroso-N-ethylurea and N-methyl-N'-nitro-N-nitrosoguanidine were equally or more active in the induction of CA and SCE in CHO cells when compared with N-NO-AAF. The cell cycle was significantly delayed by the intervention of N-NO-AAF.

2-Acetylaminofluorene↗

Curcumins as inhibitors of nitrosation in vitro.

The effects of turmeric extract and its pure yellow pigments curcumin I, II and III were tested on the nitrosation of methylurea by sodium nitrite at pH 3.6 and 30 degrees C. The nitrosomethylurea formed was monitored by checking the mutagenicity in S. typhimurium strains TA1535 and TA100 without metabolic activation. Turmeric extract as well as curcumins exhibit dose-dependent decreases of nitrosation. Curcumin III was the most effective nitrosation inhibitor among the compounds tested. The simultaneous treatment of inhibitor with nitrosation precursors was essential and pre- or post-treatment of inhibitor had no effect on the mutagenicity of nitrosomethylurea. The binding of nitrite with the inhibitors was studied at pH 3.6 and 30 degrees C. Curcumin I shows a dose-dependent depletion of nitrite ions thus making nitrite non-available for nitrosation. Curcumin I and III when tested also showed a time-dependent depletion of nitrite ions at pH 3.6 and 30 degrees C. Curcumin III has a higher affinity for nitrite ions than curcumin I.

Catechols↗

Chemoprevention of breast cancer with retinoids.

Fenretinide [N-(4-hydroxyphenyl)retinamide, 4-HPR] is an effective agent for the inhibition of N-nitroso-N-methylurea-induced breast cancer in rats. This compound has been studied extensively and proved to be safer and less teratogenic than many other retinoids. A major characteristic of 4-HPR is its ability to concentrate in the granular and fat tissue of the breast instead of in the liver. Between January and June 1986, we carried out a phase I study on 101 patients divided into four randomized groups receiving placebo and 100, 200, and 300 mg/day of 4-HPR. Patients received the drug for 6 months without any major toxic effect. This finding was confirmed by another 6-month study in which patients received a common dose of 200 mg/day. In March 1987, a phase III study was started to evaluate the effectiveness of 4-HPR in preventing contralateral primary tumors in women who had already been treated for breast cancer. If 4-HPR succeeds in preventing second primaries in breast cancer patients, it may be useful for a wider group of subjects at high risk for breast cancer. This randomized study was designed with two arms: an intervention group versus a group receiving no treatment. Patients in the intervention group will be treated with 200 mg/day 4-HPR for 5 years. Patients in the control group will not be treated. A further 2 years of follow-up is planned for both groups. Currently, 2450 patients have been recruited. We expect a total accrual of 3500 patients by the end of 1992.

Adult↗

Studies related to nitrosamide formation: nitrosation in solvent: water and solvent systems, nitrosomethylurea formation in the rat stomach and analysis of a fish product for ureas.

Nitrous acid (HNO2) was partly extracted from water by organic solvents, especially polar ones. Carbaryl was nitrosated in solvent: water mixtures most rapidly when nonpolar solvents, e.g. methylene chloride, carbon tetrachloride and hexane, were used. Under given conditions, carbaryl was nitrosated in methylene chloride:water mixtures 20 times faster than in water alone, mainly because of its insolubility in water. For ethylurea, hexylurea, ethyl N-ethyl-carbamate and aminopyrine, nitrosation by sodium sulfate-dried methylene chloride extracts of nitrous acid ('dried HNO2') was at least 88% complete after reaction for 5 seconds at 6 degrees C. Nitrosation of N-butylacetamide by the same extract proceeded more slowly, with a second-order rate constant 31,000 times greater than for nitrosation in water at pH 2. Butylacetamide was nitrosated in methylene chloride by equivalent concentrations of 'dried HNO2', dinitrogen trioxide and dinitrogen tetroxide at similar rates. Nitrosomethylurea (NMU) formation was measured radioactively in the stomach contents of rats fed [3H]methylurea (MU) and sodium nitrite. When both compounds were given in the food (100 mg MU and 4 g NaNO2/kg) and the rats were killed 3 hours later, the NMU yield was 0.46% of the MU. When the food also contained 11.5 g sodium ascorbate/kg, NMU production was completely inhibited. With 2-4 g/1 sodium nitrite in the drinking water and MU in the food, no NMU was detected. Ureas were determined in dried, salted bonito fish from Japan, by a method involving ion-exchange and paper chromatography. The fish sample contained 80 mg urea/kg, but no MU. When the fish was nitrosated at pH 1 and then denitrosated at pH 0, 25 mg MU/kg was detected. Mu identity was confirmed by mass spectrometry.

Animals↗

Biochemical pharmacology of N-acetyl-N-(methylcarbamoyloxy)-N'-methylurea (caracemide; NSC-253272).

Preclinical pharmacologic studies of caracemide [N-acetyl-N-(methylcarbamoyloxy)-N'-methylurea; CAR] have demonstrated a marked instability of this compound in the presence of either phosphate buffer (pH 7.4) or human plasma. Using [1-14C-acetyl]CAR and [3H-methylcarbamoyloxy]CAR, three CAR degradation products were identified: product A, N-(methylcarbamoyloxy)acetamide; product B: N-(methylcarbamoyloxy)-N'-methylurea; and product C: N-hydroxy-N'-methylurea. CAR degradation in human plasma was demonstrated by high-performance liquid chromatography (HPLC) to occur in a time- and temperature-dependent manner. A 30-min incubation (37 degrees) of CAR (10(-4) M) with human plasma resulted in degradation of more than 55% of parent compound; at 1 hr, more than 75% of original CAR was degraded. Incubation of [1-14C-acetyl]CAR with rat brain homogenate resulted in the formation of 14CO2. This reaction was partially inhibited by coincubation with physostigmine (10(-3) M). CAR inhibited acetylcholinesterase activity in neuroblastoma cells with an IC50 of 14 microM. In mechanism of action studies, CAR was found to inhibit ribonucleotide reductase activity but only at nine times the IC50 of hydroxyurea. In contrast to hydroxyurea, CAR was found to be non-cell-cycle phase-specific and non-cross-resistant with two CHO cell lines resistant to hydroxyurea. These data demonstrate the instability of CAR; moreover, they suggest that its mechanism of cytotoxicity is distinctly different from that of hydroxyurea and that the neurotoxicity associated with CAR administration may be caused in part by inhibition of acetylcholinesterase activity.

Acetylcholinesterase↗

Effects of food components and additives on the formation of nitrosamides.

The effects of food components and food additives on the formation of nitrosamides were studied. Citrate accelerated nitrosamide formation from nitrite and various precursors, such as ureas, urethanes, guanidines and amides. The accelerating effects of citrate on the formation of methylurea and ethylenethiourea were larger at higher pH values. The formation of most nitrosamides was not accelerated by thiocyanate, which is known to catalyze the formation of nitrosamines. Compounds having carboxyl groups tended to accelerate the nitrosation of methylurea. Nitrosation of methylurea was inhibited by thiols, polyphenols, alcohols, sorbic acid, dl-alpha-tocopherol etc., due to possibly competition for available nitrite.

Chemical Phenomena↗

Carcinogenicity of methylurea or morpholine in combination with sodium nitrite in rat multi-organ carcinogenesis bioassay.

For carcinogenic risk assessment of combinations of N-nitroso precursors in man, the effects of feeding methylurea (MU) or morpholine (Mor) plus sodium nitrite (NaNO2) were investigated using a multi-organ carcinogenesis model. In experiment 1, to initiate multiple organs, groups of 10 or 20 male F344 rats were treated with 6 carcinogens targeting different organs. Starting a week after completion of this initiation phase, animals were given 0.1% MU or 0.5% Mor in their food and/or 0.15% NaNO2 in their drinking water for 23 weeks. The induction of tumors and/or preneoplastic lesions in the forestomach and esophagus was significantly increased in the group receiving MU plus NaNO2. The numbers and areas of liver glutathione S-transferase placental form (GST-P)-positive foci were significantly elevated with MU or Mor plus NaNO2. Experiment 2 was conducted to assess formation of N-nitroso compounds in the stomach, and to detect DNA adduct generation in target organs by immunohistochemical staining. Groups of 5 or 14 animals were starved overnight, then given 0.4% MU or 2.0% Mor in the diet, or basal diet alone for 1 h. Then NaNO2 or distilled water was given intragastrically. The mean gastric N-methyl-N-nitrosourea yield in the MU plus NaNO2 group was 7700 micrograms at 2 h after combined administration. The mean N-nitrosomorpholine yield in the group given Mor plus NaNO2 was 6720 micrograms. Immunohistochemically, N7-methyldeoxyguanosine-positive nuclei were evident in the forestomach epithelium at 8 h after the combination treatment with MU plus NaNO2.

Administration, Oral↗

Leaching of pesticides through normal-tillage and low-tillage soil--a lysimeter study. I. Isoproturon.

Isoproturon is a herbicide, which was used in Denmark against grass weeds and broad-leaved weeds until 1998. Isoproturon has frequently been detected in ground water monitoring studies. Leaching of isoproturon (N,N-dimethyl-N'-(4-(1-methylethyl)-phenyl)urea) and its metabolites, N'-(4-isopropylphenyl)-N-methylurea and N'-(4-isopropylphenyl)urea was studied in four lysimetres, two of them being replicates from a low-tillage field (lysimeter 3 and 4), the other two being replicates from a normal tillage field (lysimeter 5 and 6). In both cases the soil was a sandy loam soil with 13-14% clay. The lysimetres had a surface area of 0.5 m2 and a depth of 110 cm. Lysimeter 3 and 4 were sprayed with unlabelled isoproturon while lysimeter 5 and 6 was sprayed with a mixture of 14C-labelled and unlabelled isoproturon. The total amount of isoproturon sprayed onto each lysimeter was 63 mg, corresponding to 1.25 kg active ingredient per ha. The lysimeters were sprayed with isoproturon on October 26, 1997. The lysimetres were installed in an outdoor system in Research Centre Flakkebjerg and were thus exposed to normal climatic conditions of the area. A mean of 360 l drainage water were collected from lysimeter 3 and 4 and a mean of 375 litres from lysimeter 5 and 6. Only negligible amounts of isoproturon and its primary metabolites were found in the drainage water samples, and thus no significant difference between the two lysimeter sets was shown. In a total of 82 drainage water samples, evenly distributed between the four lysimetres isoproturon was found in detectable amounts in two samples and N'-(4-isopropylphenyl)urea was found in detectable amounts in two other samples. The detection limit for all the compounds was 0.02 microg/l. 48% and 54% of the added radioactivity were recovered from the upper 10 cm soil layer in lysimeter 5 and 6, respectively, and 17 and 14% from 10-20 cm's depth. By extraction first with an aquatic CaCl2 solution 0.49% of the added radioactivity was extracted from the upper 10 cm layer in lysimeter 5. In the subsequent extraction with acetonitril, 1.19% of the added radioactivity was extracted. In lysimeter 6, upper 10 cm, 0.2% were extracted with water and 0.56% were extracted with acetonitril. Below 10 cm's depth no measurable amounts could be extracted.

Agriculture↗

Synthesis and antiarrhythmic activity of new benzofuran derivatives.

Various 5-aminobenzofuran derivatives were prepared from khellin and screened intravenously in the dog for their potential antiarrhythmic activity against ouabain-induced ventricular arrhythmia and in the Harris test. From systematic structural variations it was found that two methoxy groups in positions 4 and 7 on the benzofuran ring, a tertiary aminoethoxy side chain in position 6, and a N-methylurea group in position 5 led to the most active compounds. These were then tested orally in the Harris test in the dog. The two long-acting derivatives N-[4,7-dimethoxy-6-(2-pyrrolidinoethoxy)-5-benzofuranyl]-N'-methylurea (8j) and N-[4,7-dimethoxy-6-(2-piperidinoethoxy)-5-benzofuranyl]-N'-methylurea (8m) showed advantages when compared to quinidine and disopyramide and have been selected for further studies.

Animals↗

[The effect of the potential antiradiation agent compound HC-1539 on reparative DNA synthesis induced by the damaging action of the chemical mutagen N-nitroso-N-methylurea and radiation].

The influence of NS-1539, a potential anti-irradiation agent and indole analogues of tamoxifen on the DNA repair synthesis in the bone marrow and lymphocytes of peripheral blood was studied. It was found that the DNA repair processes from damages caused by chemical mutagen N-nitroso-N-methylurea or physical factor UV-irradiation in the bone marrow cells after the NS-1539 protection of mice as well as in lymphocytes of peripheral blood of human donors were enhanced. The enhancing of DNA repair synthesis was seen not earlier than 18 hours after the NS-1539 injection to mice in vivo or when NS-1539 was added to lymphocyte suspension in vitro and observed up to the third day. This period of time coincides with the time of formation of radioresistance of organism under the influence of this agent as well as with the time of realization effects of steroid hormones on target cells.

Animals↗