Synthesis, characterization, and immunochemical detection of O6-(carboxymethyl)-2'-deoxyguanosine: a DNA adduct formed by nitrosated glycine derivatives.

O6-(Carboxymethyl)-2'-deoxyguanosine (O6-CMdG) is formed in DNA by nitrosated glycine derivatives and appears to be nonrepairable by O6-alkylguanine transferases. O6-CMdG has been synthesized by an unambiguous route involving the introduction of a methyl glycolate moiety at C6 of a 3',5'-bis-O-(methoxyacetyl)dGuo derivative by displacement of a quinuclidinium ion. Methanolysis of the methoxyacetyl groups and calcium hydroxide-mediated hydrolysis of the methyl ester afforded the calcium salt of O6-CMdG in good yield. A similar route was used to synthesize O6-(carboxymethyl)guanosine (O6-CMGuo), which was used to prepare protein conjugates for immunization. Rabbit antisera were prepared, and a quantitative competitive ELISA was developed which showed 50% inhibition at 2 pmol of O6-CMdG/ well. O6-CMGuo was 30 times less cross-reactive (50% inhibition at 60 pmol/well), and normal nucleosides and carboxymethylated purines did not cross-react to any significant extent. The antiserum was also used to prepare reusable immunoaffinity columns which retained O6-CMdG. The binding of O6-CMdG was so strong that conditions used to elute the adduct (1 M trifluoroacetic acid) resulted in partial hydrolysis (becoming quantitative on heating) of the glycosidic bond to give O6-CMguanine which was detected by HPLC with fluorescence detection. DNA treated with azaserine (5 mmol), N-(N'-acetyl-L-prolyl)-N-nitrosoglycine (5 mmol), and potassium diazoacetate (5 mmol) afforded O6-CMdG at levels of 7.3, 393.9, and 496 mumol of O6-CMdG/mol of dG. The antiserum also recognized O6-CMdG in intact DNA.

A screening procedure for total N-nitroso contaminants in personal care products: results of collaborative studies undertaken by a CTPA Working Group.

Synopsis A modified procedure for the determination of total N-nitroso compounds in personal care products was evaluated in collaborative studies organized through the UK Cosmetic Toiletry and Perfumery Association (CTPA). The method offers a true 'totals'determination in that a solution of the whole sample is analysed. Samples are dissolved/suspended in water or aqueous THF, and nitrite/nitrite ester interferences are removed by prior treatment with sulphamic acid. The treated test solution is denitrosated in a single reaction with HBr/acetic acid, in refluxing n-propyl acetate, and 'total'N-nitroso compounds are determined in a chemiluminescence reaction of the released nitric oxide with ozone. The use of a propyl acetate denitrosation solvent and of higher concentrations of HBr have improved both the sensitivity (routine limit of determination at 10 mugkg(-1)) and water tolerance of the method. The method was shown to recover N-nitrosamines quantitatively, and to be sufficiently repeatable and reproducible to be used as a screening technique for N-nitroso compounds in personal care products.

Reduction of nitrosamines in cosmetic products.

Summary From initial rate studies of morpholine nitrosation in aqueous media by nitrite ion in the presence of formaldehyde at pH 5-7 and 25 degrees C, four potential pathways are revealed for the concurrent formation of nitrosamine contaminants in cosmetic and toiletry products. Three of the pathways involve conventional electrophilic nitrosation by XNO reagents of both neutral amine and N-hydroxymethylamine compounds obtained by prior reaction with formaldehyde. The fourth pathway involves a nucleophilic reaction by nitrite ion with the iminium ion derived from N-hydroxymethylamine. For morpholine, reaction via XNO reagents is substantial at pH 5 only, whereas the iminium ion pathway is pre-eminent at pH 7. The concurrent formation of nitrosamines by mechanistically different pathways implies that combinations of nitrosation inhibitors are necessary to minimize contamination of cosmetic and toiletry products. For these different pathways, novel inhibitory compounds are described which fulfil the usual acceptance criteria for cosmetic and toiletry materials. The efficacy of these compounds is assessed against N-nitrosomorpholine formation in the presence of formaldehyde. These include erythorbate, ascorbate, pentanedione and pyranone compounds for the XNO pathways (pH 6) and neutral organic and inorganic salts for the iminium ion pathways (pH 7). Preliminary results for both a bath gel and a cream-base formulation deliberately adulterated with morpholine, diethanolamine, nitrite and a preservative which releases low levels of formaldehyde on decomposition show better than 90% inhibition of nitrosamines by selected pairs of inhibitor compounds on storage at 40 degrees C over several months. This novel technology is the subject of a worldwide patent application.

Chemistry and biology of nitrosated peptides.

All peptides undergo nitrosation at the terminal primary amino group to generate a diazopeptide and at the peptide N-atoms to produce an N-nitrosopeptide. With acidified nitrite, diazopeptides usually form more readily than N-nitrosopeptides, but quickly decompose. Diazopeptides form rapidly at neutral pH from gaseous nitrogen oxides and are relatively stable under these conditions. N-nitrosopeptides are more stable than diazopeptides at both acidic and neutral pH. Because of their availability as substrates, peptides are probably involved in endogenous nitrosation associated with the diet and from the inhalation of nitrogen oxides as in tobacco smoking. Both diazopeptides and N-nitrosopeptides are cytotoxic, but a causal role for these compounds in human cancer has yet to be demonstrated.

The donor specificity and kinetics of the hydrolysis reaction of gamma-glutamyltransferase.

The donor specificity of the hydrolytic reaction of gamma-glutamyltransferase [5-glutamyl)-peptide:amino-acid 5-glutamyltransferase, EC 2.3.2.2) has been studied by the use of specifically synthesised gamma-glutamyl substrates. It was found that a wide variety of gamma-glutamylated adducts were hydrolysed by the enzyme. The structure of the adduct was relatively unimportant for donor specificity and the enzyme appears to 'recognise' the gamma-glutamyl portion of the donor molecule. In particular the alpha-amino group and the free proton of the gamma-peptide bond appear to be essential for donor activity. The Vmax of hydrolysis increased proportionally to the electron-withdrawing capacity of the adduct moiety. The rate of formation of gamma-glutamyl-enzyme intermediate was therefore dependent upon the structure of the adduct of the gamma-glutamyl donor. The results suggest that the enzyme shows little specificity beyond that for gamma-glutamyl amides and there is therefore no reason to postulate the presence of a specific glutathione-binding site.

Formation of diazopeptides by nitrogen oxides.

A versatile synthesis of diazopeptides is reported and several new compounds described. It is shown that diazopeptides form readily from gaseous nitrogen dioxide in both neutral buffers and human blood. In these media, diazopeptides are sufficiently stable (half-time 0.5-30 h) to qualify as potential circulating carcinogens.

Peptide nitrosation in dilute acid.

Rates and products are reported for the nitrosation of simple dipeptides (glycylglycine, its ethyl ester and N-acetylglycylglycine) in dilute acid at 37 degrees C. The results suggest that conversion to a diazo derivative (which rapidly decomposes) is the most likely outcome of the gastric nitrosation of small proteins and peptides.

Bacterially mediated N-nitrosation reactions and endogenous formation of N-nitroso compounds.

Results are presented demonstrating some factors that affect the kinetics of bacterially mediated N-nitrosation reactions. Two groups of bacteria, differing in their nitrate/nitrite metabolism, are contrasted. These findings are discussed in relation to a role for bacteria in endogenous N-nitrosation reactions.

Mutagenic properties of N-nitrosopeptides in mammalian cell culture assays.

Two N-nitrosopeptides, N-(N-acetyl-L-prolyl)-N-nitrosoglycine and N-(N-acetylvalyl)-N-nitrosoglycine, were investigated for genetic toxicity towards mammalian cells using an established line of Chinese hamster ovary cells (CHO-K1-BH4). Observations were made on three indices of genetic damage, namely chromosome aberrations, sister chromatid exchange and induction of thioguanine-resistant variants. Treatment of cells with either compound resulted in dose-dependent increases in all indices, indicating that both compounds are direct-acting mutagens.

The mutagenic properties of N-nitrosopeptides in the Ames test.

N-(N-Acetyl-L-prolyl)-N-nitrosoglycine (APNG) and N-(N-acetylvalyl)-N-nitrosoglycine (AVNG) are shown to exert mutagenic activity in the Salmonella/mammalian microsome mutagenicity (Ames) test. Positive responses are apparent for base-pair substitution mutation-detecting strains (TA1535, TA100 and TA102) both with and without the addition of S9-mix. It is concluded that both APNG and AVNG are direct-acting mutagens.

Nitrosation of peptides.

The synthesis and characterization of N-(N-acetylprolyl)-N-nitroso-glycine, the first authentic N-nitrosopeptide, is described, and its stability under various conditions is reported. In acidic media, denitrosation and deamination (hydrolysis) occur concurrently, whereas in neutral and alkaline solutions, only deamination occurs. The rates of formation and decomposition of some unprotected N-nitrosopeptides in strong acid are also reported. Conditions for the formation of such compounds in the gastric tract are discussed, together with their potential involvement in human cancer.

Amine nitration and nitrosation by gaseous nitrogen dioxide.

Reaction of N-methylaniline in solution with ca. 100,000 ppm gaseous NO2 leads to significant amounts of ring- and N-nitro products, in addition to N-methyl-N-nitrosoaniline. Reaction of heterocyclic amines in solution with 5-1000 ppm gaseous NO2 gives significant amounts of N-nitrosamines and much higher amounts of N-nitramines than those obtained from concentrated gaseous NO2. The results are interpreted as evidence that N-nitrosation proceeds via an unsymmetrical ON-ONO2 dimer, whereas N-nitration may involve a free-radical process involving NO2, itself.

Formation of N-nitrosamines and N-nitramines by photolysis.

N-Nitrosamines are generated by the photolysis of neutral aqueous solutions of nitrite salts and heterocyclic amines. With nitrate salts, photolysis produces a mixture of N-nitrosamines and N-nitramines. The reactions occur with low (5 mmol/l) reagent concentrations under very mild conditions and are faster than the concurrent decomposition of N-nitrosamines and N-nitramines by photolysis. Furthermore, both N-nitrosamines and N-nitramines effect transnitrosation reactions under mild neutral conditions when photolysed in the presence of other amines.

Endogenous synthesis of volatile nitrosamines: model calculations and risk assessment.

A kinetic model for estimating the gastric synthesis of N-nitroso compounds and a method for estimating the risk from exposure to volatile nitrosamines are described. A tentative calculation of the possible risks for human gastric cancer, based on extrapolating from literature data on the induction of tumours in rats, suggests that the endogenous formation of non-volatile N-nitroso compounds is more important than that of volatile nitrosamines.

Protective effect of a new antioxidant on acute hepatotoxicity caused by morpholine plus nitrite in rats.

Bis(2,2-dimethyl-4-methane sulphonic acid sodium salt-1,2-dihydroquinoline)-6,6'-methane (MTDQ-DA), a new, non-toxic, water soluble antioxidant, is shown to inhibit liver necrosis induced in rats by N-nitrosomorpholine (N-MOR), itself formed in vivo following the administration simultaneously of morphine (MOR) and sodium nitrite (NaNO2).

A comparative electron microscope study of early changes in rat liver induced by N-nitrosopiperidine and 2,2',6,6'-tetramethyl-N-nitrosopiperidine.

Inbred male Leeds rats were administered either the liver carcinogen N-nitrosopiperidine or the non-carcinogen 2,2',6,6'-tetramethyl-N-nitrosopiperidine in their drinking water at a concentration of 0.02%. Treatment was continued until the animals were killed, at 12 or 28 days, when their hepatic tissues were removed and examined by electron microscopy. Both compounds induced glycogen depletion, cytoplasmic fat accumulation and proliferation of the smooth endoplasmic (ER). In addition, NPIP induced mitochondrial and bile canalicular changes and marked reduction and alteration of the rough ER. The probable relationships of the observed fine structural changes to the metabolism, toxicity and carcinogenicity of these heterocyclic N-nitrosamines are discussed.

New pathways for the rapid formation of N-nitrosamines under neutral and alkaline conditions.

Ethylene glycol, several carbohydrates (sugars) and alkanolamines influence the formation of carcinogenic N-nitrosamines in neutral and alkaline aqueous solutions at 25 degrees C in presence of dissolved nitrosyl gases. These compounds either catalyse or inhibit the reactions (depending on the experimental conditions and reagent reactivities) by forming a nitrite ester intermediate, which reacts readily with secondary amines. The reactions may explain the origin of some N-nitrosamines in vivo and in consumer products, particularly those originating from NOX pollutants. N-Nitrosamines are also formed at ambient temperatures by the gamma-radiolysis of neutral aqueous solutions of either NaNO2 or NaNO3 and secondary amines. With NaNO3, N-nitroamines are in accompanying product. These reactions are considered to proceed via N2O3 and N2O4 intermediates, generated from NaNO2 and NaNO3, respectively.