Nitrite, nitrosamines, and cancer.

Carcinogenic N-nitroso compounds are formed from the reaction of naturally-occurring amines and nitrites that may be added to foods or produced by bacterial reduction of nitrate. N-Nitroso compounds can be produced during processing, storage and preparation of foods and in the mammalian stomach. Factors that influence the rates of nitrosation reactions include pH, temperature, catalysts, and inhibitors. Predictions of the extent of nitrosation are complicated by these factors and ultimately the amounts and types of N-nitroso compounds present must be determined by direct analysis. Methods for detection and estimation of volatile nitrosamines are available and low levels (parts per billion) have been found in some cured meat and fish products. General methods for detection of all N-nitroso compounds are not available yet, but are under development. Evaluation of the risk to human populations from these compounds is difficult in the absence of more comprehensive data on their environmental distribution.     

Vasodilator actions of several N-nitroso compounds

Recent studies have shown that N-nitroso compounds can activate arterial guanylate cyclase and relax isolated arterial smooth muscle; however, the effects of these substances on the cardiovascular system in the anesthetized cat are unknown. The present study was undertaken to compare the effects of several nitrosoguanidines and a nitrosamine, N-nitrosodimethylamine, on arterial guanylate cyclase activity, isolated arterial smooth muscle tone, and systemic vascular resistance in the anesthetized cat. Intravenous injections and infusions of the nitrosoguanidines glyceryl trinitrate (GTN) and sodium nitroprusside (SNP) decreased systemic arterial pressure. During intravenous infusion of the nitrosoguanidines GTN and SNP, cardiac output was unchanged at the peak of the decrease in aortic pressure, indicating that the nitrosoguanidines GTN and SNP both reduced systemic vascular resistance. In addition, intraarterial injections of the nitrosoguanidines produced dose-dependent decreases in perfusion pressure in the feline mesenteric vascular bed perfused at constant flow. These substances were potent relaxants of isolated arterial smooth muscle and markedly activated arterial guanylate cyclase. In contrast, N-nitrosodimethylamine was devoid of vasodilator activity in vivo and exerted only minimal effects on isolated arterial smooth muscle tone or on arterial guanylate cyclase activity. The present data demonstrate a relationship between guanylate cyclase activation and arterial smooth muscle relaxation and suggest that the vasodilator effects on resistance vessels in vivo in response to selected N-nitroso compounds may involve such a mechanism. Although the significance of the presently reported cardiovascular responses to N-nitroso compounds is uncertain, N-nitroso compounds may represent a previously unrecognized class of substances which can be formed in the body and which possess marked vasodilator activity. It is possible that this vasodilator activity may involve the relaxation of vascular smooth muscle through activation of guanylate cyclase.     

Mechanisms of inhibition of N-nitroso compounds-induced mutagenicity

In this review we describe the mechanisms of the inhibitory effects of various chemical agents towards the mutagenicity of N-nitroso compounds, including direct-acting mutagens such as N-nitroso derivatives of alkylureas, alkylnitroguanidines and alkylurethanes, and promutagenic nitrosamines. Possible mechanisms by which the inhibitors may exert their effects outside and inside the target cells include chemical and enzymatic deactivation of the mutagen, inhibition of metabolic activation of nitrosamines, scavenging mutagenic products, inhibition of cellular uptake, induction of detoxifying mechanisms, protecting nucleophilic centers in DNA and modulating DNA repair.     

Characterization of cerebellar guanylate cyclase using N-methyl-N'-nitro-N-nitrosoguanidine. Presence of two different types of guanylate cyclase in soluble and particulate fractions

Some characteristics of guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) in subcellular fractions prepared from rat cerebellum have been analyzed on the basis of responsiveness to N-methyl-N'-nitro-N-nitrosoguanidine and inhibitors related to N-nitroso compounds. The enzyme in 100 000 X g supernatant and crude mitochondrial (P2) fractions were differently activated (11- and 2.5-fold, respectively) by N-methyl-N'-nitro-N-nitrosoguanidine. The soluble fraction obtained by hypo-osmotic treatment and subsequent recentrifugation of the P2 (P2-soluble) contained a significantly higher total guanylate cyclase activity than that of the starting material (P2). The P2-soluble fraction also exhibited a lower responsiveness (1.5-fold) to N-methyl-N'-nitro-N-nitrosoguanidine than that found in the P2. The membrane fraction prepared from the P2 (P2-membrane) had no response to N-methyl-N'-nitro-N-nitrosoguanidine. Hemoglobin and vitamin A derivatives significantly inhibited both N-methyl-N'-nitro-N-nitrosoguanidine-activated 100 000 X g supernatant and basal P2-soluble enzyme activities, without effect on the basal activities in 100 000 X g supernatant and P2-membrane fractions. The present results suggest that two different types of guanylate cyclase may be present in rat cerebellum in terms of the responsiveness of N-nitroso compounds, and P2-soluble guanylate cyclase seems to be activated endogenously through a mechanism similar to the action of N-methyl-N'-nitro-N-nitrosoguanidine.     

Food-borne amines and amides as potential precursors of endogenous carcinogens

This paper reviews the experimental results of our research in the past several years and other related papers that have been directed toward the occurrence, biotransformation and epidemiological significance of carcinogenic N-nitroso compounds in biosphere. Endogenous carcinogens are a group of cancer-causing compounds produced in vivo from harmless precursors. This category has been exemplified by the well-known carcinogens, N-nitroso compounds. The significance of naturally occurring amines and amides as precursors of carcinogenic N-nitroso compounds in vivo and their implication in the incidence of human cancer have been investigated and emphasized. Extremely high levels of trimethylamine-N-oxide and dimethylamine were detected in squids and other seafoods. More than 90% of trimethylamine-N-oxide were converted to dimethylamine and trimethylamine on pyrolysis. Low levels of dimethylamine and methylamine were also detected in the fermented soybean products, wines and sauces. Both dimethylamine and trimethylamine are excellent precursors of dimethylnitrosamine. Several naturally occurring aromatic amines especially 2-carboline derivatives such as harman, norharman, harmaline, harmalol, harmine and harmol are mutagenic and become more mutagenic to Salmonella typhimurium after nitrosation. Appreciable amounts of piperidine were detected in the popular spice white and black pepper powders. Under acidic condition, piperidine reacts readily with nitrite to form carcinogenic N-nitroso-piperidine. N-Nitrosophenacetin was formed from the reaction of nitrite with the amide drug phenacetin. This new compound showed strong mutagenicity to Salmonella typhimurium and Sarcina lutea and strong teratogenic activity to Leghorn chicken embryos. Studies have shown that the majority of N-nitroso compounds in the body come from in vivo conversion. Most investigators believe that this endogenous pool of N-nitroso compounds may prove to be a major exposure route in man. The presence of naturally occurring amines and amides in the diet then becomes one of the crucial limiting steps in the formation of endogenous N-nitroso compounds in vivo.     

N-Nitroso compounds in the diet.

N-Nitroso compounds were known almost 40 years ago to be present in food treated with sodium nitrite, which made fish meal hepatotoxic to animals through formation of nitrosodimethylamine (NDMA). Since that time, N-nitroso compounds have been shown in animal experiments to be the most broadly acting and the most potent group of carcinogens. The key role of nitrite and nitrogen oxides in forming N-nitroso compounds by interaction with secondary and tertiary amino compounds has led to the examination worldwide of foods for the presence of N-nitroso compounds, which have been found almost exclusively in those foods containing nitrite or which have become exposed to nitrogen oxides. Among these are cured meats, especially bacon-and especially when cooked; concentrations of 100 micrograms kg(-1) have been found or, more usually, near 10 micrograms kg(-1). This would correspond to consumption of 1 microgram of NDMA in a 100-g portion. Much higher concentrations of NDMA (but lower ones of other nitrosamines) have been found in Japanese smoked and cured fish (more than 100 micrograms kg(-1)). Beer is one source of NDMA, in which as much as 70 micrograms l(-1) has been reported in some types of German beer, although usual levels are much lower (10 or 5 micrograms l(-1)); this could mean a considerable intake for a heavy beer drinker of several liters per day. Levels of nitrosamines have been declining during the past three decades, concurrent with a lowering of the nitrite used in food and greater control of exposure of malt to nitrogen oxides in beer making. There have been declines of N-nitroso compound concentrations in many foods during the past two decades. The small amounts of nitrosamines in food are nonetheless significant because of the possibility-even likelihood-that humans are more sensitive to these carcinogens than are laboratory rodents. Although it is probable that alkylnitrosamides (which induce brain tumors in rodents) are present in cured meats and other potentially nitrosated products in spite of much searching, there has been only limited indirect evidence of their presence.     

Several known indole compounds are not important precursors of direct mutagenic N-nitroso compounds in green cabbage

In this study we investigated the role of indole-3-acetonitrile, indole-3-carbinol, indole and tryptophan in the formation of N-nitroso compounds in green cabbage extracts. Green cabbage extracts were separated by gel permeation chromatography. Fractions were treated with nitrite, tested for mutagenicity and analysed for total N-nitroso content. Fractions in which spiked indole-3-acetonitrile, indole-3-carbinol, indole and tryptophan eluted appeared to be low in mutagenic activity and contained relatively small amounts of N-nitroso compounds. To detect indole compounds other than the ones used in the gel permeation chromatography experiments, high-performance liquid chromatography and gas chromatography-mass spectrometry analyses were performed of green cabbage extracts. Indole-3-carboxaldehyde was found to be the most commonly occurring indole compound, but it did not show direct mutagenic activity upon nitrite treatment. Indole-3-acetonitrile was the second most common compound; although it was mutagenic after nitrite treatment, its contribution to the mutagenicity of nitrite-treated green cabbage was roughly estimated to be only 2%. No other indole compounds were detected. From this study we conclude that neither the tested indole compounds nor indole-3-carboxaldehyde play a significant role in the formation of direct mutagenic N-nitroso compounds in nitrite-treated green cabbage extracts.     

N-nitrosoanilines: a new class of caspase-3 inhibitors

Caspases are a family of cysteine proteases activated during apoptosis. In cultured human endothelial cells, physiological levels of NO prevent apoptosis and interfere with the activation of the caspase cascade. Previous studies have demonstrated that NO inhibits the activity of caspase-3 by S-nitrosylation of the enzyme. In this study, the inhibitory effect of a new class of NO donors. N-nitrosoaniline derivatives, were examined against caspase-3. Initially eight small molecule inhibitors bearing N-nitroso moieties were assayed. It was found that the presence of an electron-donating group on the phenyl ring led to better inhibitory potency, a trend consistent with the results from the previous papain studies. Based on the analysis of the enzyme and substrates' structures, two peptidyl N-nitrosoaniline inhibitors [Ac-DVAD-NNO (1) and Ac-DV-AMO (2)] were designed and synthesized. Both compounds exhibited enhanced inhibitory potency against caspase-3.     

Mutagenesis by N-nitroso compounds in Salmonella typhimurium TA102 and TA104: evidence for premutagenic adenine or thymine DNA adducts

Mutagenesis induced by the N-nitroso compounds: N-nitrosomethylurea, N-nitrosoethylurea, N-nitrosodi-n-propylamine and N-nitrosopyrrolidine was measured in Salmonella typhimurium TA100, TA102 and TA104. TA100 detects damage mainly at G-C base pairs while TA102 and TA104 can detect damage at A-T base pairs. In general all strains were similarly sensitive, except that TA104 was much less sensitive to high doses of N-nitroso-N-methylurea. In TA104 a significant percentage of the revertants induced by all agents except NMU resulted from point mutations at A-T base pairs, indicating that adenine or thymine DNA adducts are important premutagenic adducts formed by certain N-nitroso compounds.     

Prophage Induction in Lysogenic Escherichia coli with N-Nitroso Compounds and Derivatives

Prophage induction in lysogenic Escherichia coli W1709 (iota) was determined for 29 N-nitroso compounds, 13 of their denitrosated derivatives, and 7 hydroxylamino and hydrazino analogues of nitrosamines. Minimal inducing concentrations of 0.1 to 2.0 mug/ml were demonstrated for eight nitrosamidines, and concentrations of 0.5 to 25.0 mug/ml were shown for six nitrosamides. Weak inducing activities were found with N,N-diethylhydroxylamine oxalate and N-methyl-N-phenylhydrazine sulfate, derivatives of inactive N-nitrosodiethylamine and N-nitrosomethylphenylamine, respectively. Inactive compounds including N-methyl-N-nitroso-p-toluenesulfonamide, 11 nitrosamines, 3 N, N'-dialkyl substituted-N-nitrosoureas, 13 denitrosated derivatives, and 5 hydroxylamino and hydrazino analogues of nitrosamines are listed. Since 7 of the 14 prophage-inducing nitrosamidines and nitrosamides reported thus far have carcinostatic activity in rodent tumor systems, it is concluded that the induction test may provide a useful screen for the detection of potential antitumor compounds. The induction test may also be useful for the detection of responsive N-nitroso compounds which may be potential toxicological hazards in the environment since, of the six active nitrosamides, five have already been reported to produce mutagenic and carcinogenic effects, four produce chromosomedamaging effects, and two produce teratogenic effects. Use of the prophage induction system for detection of biologically active intermediates formed by N-nitroso compounds under physiological conditions is considered.     

Effect of histamine H2-receptor antagonist therapy on the mutagenic activity of gastric juice

There is concern at present that treatment with histamine H2-receptor antagonists might promote the development of gastric cancer by producing conditions which favour intragastric formation of N-nitroso compounds. If H2-receptor antagonist therapy causes increased intragastric levels of N-nitroso compounds, an issue not yet resolved by analytical studies, corresponding changes in the mutagenic activity of gastric juice might be anticipated. In this study mutagenic activity and pH were measured in fasting gastric aspirate from 18 peptic ulcer patients before and during the final week of therapy with ranitidine (n = 10) or cimetidine (n = 8). Mutagenic activity was assessed using Salmonella typhimurium TA98 and TA100 in a modified pre-incubation "fluctuation" test. No significant change in mutagenic activity was detected after therapy. Of 15 patients found to have significant mutagenic activity in their fasting gastric juice before treatment, 14 remained mutagenic following treatment. Mutation frequencies (sum of positive wells in duplicate 96-well microtitre plates, mean +/- SD) for TA98 and TA100 were respectively, 20 +/- 34 and 100 +/- 64 before compared with 10 +/- 6 and 102 +/- 65 after therapy (p greater than 0.05). Changes in mutagenic activity were similar in both treatment groups and unrelated to duration of therapy, changes in gastric pH or ulcer healing. In vitro, neither cimetidine in aqueous solution, nor gastric juice preincubated with cimetidine showed significant mutagenic activity. These results provide no evidence that increased intragastric levels of genotoxic chemicals, such as N-nitroso compounds, occur during H2-receptor antagonist therapy.     

Methylation of DNA by three N-nitroso compounds: evidence for sequence specific methylation by a common intermediate

Methylation in vitro of calf thymus DNA, a supercoiled plasmid, poly(dG).poly(dC), and poly(dGdC).poly(dGdC) by N-nitroso(acetoxy-methyl)methylamine and N-nitroso(acetoxybenzyl)methylamine in the presence of esterase, and by N-nitrosomethylurea was investigated. Although there were differences in the amounts of 7-methylguanine and O6-methylguanine formed in the various DNA substrates, the methylation pattern was the same for each of these methylating agents. The three compounds reacted identically when methylation of a portion of a 345 bp restriction fragment of the plasmid pBR322 was examined at nucleotide resolution by a sequencing assay. They also showed a tendency to react preferentially with particular guanines. These data suggest that the three N-nitroso compounds methylate DNA via a common intermediate such as the methyl diazonium ion, which exhibits some sequence specificity.     

Photolysis of nitrosamines and nitrosamides at neutral pH: a spin-trap study

A model system has been used to study the types of radicals formed on denitrosation of N-nitroso compounds. Free radicals were formed at room temperature (22 degrees-23 degrees C) and neutral pH by photolytic cleavage of N-nitroso bonds and were partially characterized following their addition to the spin traps 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and N-tert-butyl-alpha-phenyl-nitrone (PBN). Carbon-centered radical adducts were obtained during nitrosamine photolysis and nitrogen-centered radical adducts during nitrosamide photolysis. Since both the nitrosamines and nitrosamides initially form nitrogen-centered radicals on photolysis, a secondary reaction or rearrangement must occur after initial N-nitroso bond cleavage in the nitrosamines. Mechanisms are proposed to account for these results.     

Synthesis of N-substituted N-nitrosohydroxylamines as inhibitors of mushroom tyrosinase

A series of N-substituted N-nitrosohydroxylamines including six new compounds were synthesized and examined for inhibition of mushroom tyrosinase. Corresponding hydroxylamines were reacted with n-butyl nitrite to give substituted nitrosohydroxylamines as their ammonium salt. The N-substituted hydroxylamines were prepared from the primary amines via the oxaziridine, or from the carbonyl compounds via the oxime. Most of the nitrosohydroxylamines tested inhibited mushroom tyrosinase. Among them, N-cyclopentyl-N-nitrosohydroxylamine exhibited the most potent activity (IC(50)=0.6 microM), as powerful as that of tropolone, one of the most powerful inhibitors. As removal of nitroso or hydroxyl moiety, the enzyme inhibitory activity was completely diminished. Both N-nitroso group and N-hydroxy group were suggested to be essential for the activity, probably by interacting with the copper ion at the active site of the enzyme. Lineweaver-Burk plotting showed that cupferron was a competitive inhibitor but that N-cyclopentyl-N-nitrosohydroxylamine was not.     

Nitrosamine carcinogenicity: a quantitative Hansch-Taft structure-activity relationship

Statistically significant correlations have been demonstrated between carcinogenic activity, water-hexane partition coefficients and electronic factors for an extensive series of N-nitroso compounds. Electronic factors were expressed by the Taft sigma* values of substituents on the carbon atoms alpha to the N-nitroso group. Such correlations indicate that transport of the carcinogen to its active site has an important effect on its potency. The correlations also implicate reactivity at the alpha-carbon in the determination of carcinogenic activity and point out various structural types which do not follow the general rule.     

Endophytic fungi from medicinal plants: a treasure hunt for bioactive metabolites

Endophytic fungi are ubiquitous organisms found in the plants, residing intercellular or intracellular, at least for a portion of their lives without causing apparent symptoms of infection. Almost all plants are known to harbor endophytes. The choice of the plant to be used for exploring endophytes for bioactives is important. Therefore, medicinal plants which are known to be used since centuries as an alternative source of medicine, are a valuable source for bioprospecting endophytes. Nevertheless, due to many reasons there is a dire need for novel resources for novel drugs which can be an answer to many deadly diseases. It is in this context that the present review was envisaged. The review reveals the importance of endophytic fungi from medicinal plants as a source of bioactive and chemically novel compounds. The bioactive metabolites produced by endophytic fungi originate from different biosynthetic pathways and belong to diverse structural groups such as terpenoids, steroids, quinones, phenols, coumarins etc. Endophytes therefore, represent a chemical reservoir for new compounds such as, anticancer, immunomodulatory, antioxidant, antiparasitic, antiviral, antitubercular, insecticidal etc. for use in the pharmaceutical and agrochemical industries. Although, efforts have been made to accommodate as many examples as possible but the depth of the subject is so vast that it cannot be covered in one single review. This in itself speaks of the fact that endophytic fungi from medicinal plants is indeed a treasure worth searching. In the present review only some selected examples have been covered.     

Selective photo-reduction of N-nitroamines combined with micellar electrokinetic chromatography and laser fluorimetric detection

N-nitroso compounds (NOC) are potent carcinogens. Reliable methods for the analysis of volatile carcinogenic NOC are well established; however selective and sensitive methods for routine analysis of thermally unstable, ionic or non-volatile NOC are still needed. For this purpose, a method based on micellar electrokinetic chromatography (MEKC) with laser induced fluorescence (LIF) detection is described for the simultaneous determination of a broad range of N-nitroso compounds. In this procedure, the nitroso group is photolytically cleaved from the NOC to yield the corresponding amine. The amines are then derivatized with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), identified and quantified using MEKC-LIF. For the standard mixture of NOC, this method has good sensitivity and a large dynamic range. The detection limit provided by the method is 9 ppb for N-nitrosopyrrolidine.     

Characterization of a CHO variant in respect to alkylating agent-induced biological effects and DNA repair

From the Chinese hamster ovary line CHO-9 a resistant variant, Cl 3, was isolated after treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Cl 3 cells were much more resistant to the cytotoxic effects of MNNG (D10 of 1.8 microgram/ml MNNG as compared to 0.23 microgram/ml for parental line) and other methylating N-nitroso compounds, but they had the same sensitivity to various other alkylating agents. MNNG was equally effective in sensitive parent line and resistant variant in inducing sister-chromatid exchanges (SCEs) and mutations to 6-thioguanine resistance. The increased resistance of Cl 3 was not due to reduced cellular uptake of MNNG, to a more efficient repair of methylated purine bases, or to differences in MNNG-induced inhibition of DNA synthesis. It is concluded that the resistant variant has some unknown tolerance mechanism which alters the cytotoxic, but not the SCE- and mutation-inducing effects of methylating N-nitroso compounds.     

Carcinogenic N-nitrosamines in the diet: occurrence, formation, mechanisms and carcinogenic potential

Nitrosamines form a large group of genotoxic chemical carcinogens which occur in the human diet and other environmental media, and can be formed endogenously in the human body. N-Nitroso compounds can induce cancer in experimental animals. Some representative compounds of this class induce cancer in at least 40 different animal species including higher primates. Tumours induced in experimental animals resemble their human counterparts with respect to both morphological and biochemical properties. Extensive experimental, and some epidemiological data suggest that humans are susceptible to carcinogenesis by N-nitroso compounds and that the presence of these compounds in some foods may be regarded as an aetiological risk factor for certain human cancers including cancers of the oesophagus, stomach and nasopharynx.     

Synthesis of pyrazolo[4,3-e][1,2,4]triazine sulfonamides,novel Sildenafil analogs with tyrosinase inhibitory activity

Tyrosinase is a multifunctional, glycosylated and copper-containing oxidase which catalyzes the first two steps in mammalian melanogenesis and is responsible for enzymatic browning reactions in damaged fruits during post-harvest handling and processing. Neither hyperpigmentation in human skin nor enzymatic browning in fruits are desirable. These phenomena have encouraged researchers to seek new potent tyrosinase inhibitors for use in foods and cosmetics. This article surveys tyrosinase inhibitors, newly discovered from natural and synthetic sources. The inhibitory strength is comparable to that of the standard inhibitor kojic acid. Also their inhibitory mechanisms are discussed. The new obtained compounds were also tested as PDE5 inhibitors and did not show significant inhibitory effect.