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.     

International Commission for Protection against Environmental Mutagens and Carcinogens. ICPEMC Working Paper No. 15/6. Effect of ethanol on nitrosamine metabolism and distribution. Implications for the role of nitrosamines in human cancer and for the influence of alcohol consumption on cancer incidence

Alcohol consumption is associated with an increase in the incidence of cancers of several sites, including oesophagus, larynx and mouth. The mechanism of the induction of cancer by alcohol is not clear. Humans are exposed to a variety of carcinogenic N-nitroso compounds. Ethanol changes the pharmacokinetics of nitrosamines in rats particularly by decreasing the ability of the liver to metabolize them. A hypothesis is put forward that the influence of alcohol on human cancer is mediated by its effect on the metabolism and distribution of nitrosamines from the diet, from tobacco smoke and from endogenous synthesis.     

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.     

Formation of mutagenic N-nitroso compounds from the pesticides prometryne, dodine and carbaryl in the presence of nitrite at pH 1

Environmental chemicals including pesticides carrying secondary and tertiary amino groups are suggested to be a health hazard to man since potentially carcinogenic nitroso compounds may be formed in the presence of nitrite at low pH values resembling conditions in the human stomach. Nitrosation of the isopropylamino-triazine Prometryne, the n-dodecyl guanidine Dodine and the N-methylcarbamate carbaryl was investigated in the presence of HCl and acetic acid at pH 1 and excess sodium nitrite for 4 h at 37 degrees C. The reaction products were extracted with CCl4 and were analyzed qualitatively and quantitatively by infrared spectroscopy, nuclear-resonance spectrometry, GC/mass spectrometry and by spectrophotometry. All compounds investigated formed N-nitroso derivatives in the following yields: carbaryl 67%, Dodine 12% and Prometryne 14%. The N-nitroso derivatives per se were not or only slightly mutagenic to Escherichia coli K12 or Salmonella typhimurium TA 1538. However, significantly increased mutation frequencies were seen after metabolic activation by mouse-liver microsomes. These results add to the observations that among environmental chemicals not only those containing methyl- or ethyl-substituted amino groups form potentially carcinogenic nitroso derivatives but also those with iso-propylamino groups as well as alkyl-substituted guanidine derivatives.     

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.     

Quantum chemical studies of methylbenz[a]anthracenes: Metabolism and correlations with carcinogenicity

In the context of the bay region, K-region and radical cation hypotheses for polycyclic aromatic carcinogens, molecular properties were calculated for fourteen methyl derivatives of benz[a]anthracene (BA) related to (1) intrinsic substrate reactivities towards activating and detoxifying metabolism and (2)-the stabilities of the putative carbocation ultimate carcinogens. Allvalence electron methods were used, avoiding the inherent difficulties found in the π-electron methods. The calculated substrate reactivities were found to predict major metabolites sucessfully, supporting the validity of their use in attempted correlations with observed carcinogenic potencies. Positive correlations were found between observed carcinogenic potencies and (1) the reactivities of the parent polycyclic aromatic hydrocarbons (PAH) towards the initial distal bay region epoxidation and (2) the stabilities of the diol epoxide carbocations. This latter correlation holds when both the methyl derivatives of BA and previously studied unsubstituted PAH are considered together, indicating its potential usefulness as a screening parameter for carcinogenic activity.     

Effect of N-methyl-N'-nitro-N-nitrosoguanidine on amino acid incorporation into mucosal protein of rat stomach.

Administration of the carcinogenic N-nitroso compound, N-methyl-N'-nitro-N-nitrosoguanidine in drinking water (0.5 mg/mL) to male Wistar rats for 1 week caused impairment of in vivo and in vitro incorporation of [14C]leucine into stomach mucosal protein. This impairment gradually returned to normal after 4 weeks. Uptake of [14C]leucine into mucosal protein was significantly inhibited after in vitro treatment of stomach mucosa with the carcinogen. Addition of the N-nitroso compound in a cell-free system using postmitochondrial supernatant prepared from stomach mucosa also showed inhibition of amino acid incorporation. Using a more defined system consisting of purified polyribosome from stomach mucosa and pH 5 enzyme fraction derived from liver it was further demonstrated that the carcinogen purturbed protein synthesizing ability of polyribosome, under both in vivo and in vitro treatment conditions. In these respects this carcinogen has similar action on the target tissue of stomach as in the liver, although the in vivo effect may be related more to toxicity than carcinogenicity.     

Mutagenicities of nitrosated carboline derivatives

Food-borne amines have been considered as the potential precursors of endogenous carcinogenic N-nitroso compounds in humans. A compound which yields a direct mutagen after nitrite treatment was isolated from soy sauce and was identified as 1-methyl-1,2,3,4-tetrahydro-2-carboline-3-carboxylic acid (MTCA) (Wakabayashi, et al., 1983). The mutagenicities of other carboline derivatives such as harman, norharman, harmaline, harmalol, harmine, and harmol were studied. Like MTCA, the nitrosated carboline derivatives showed higher mutagenic activity as compared to their corresponding parent compounds. The demethylated analogue of MTCA, 1,2,3,4-tetrahydro-2-carboline-3-carboxylic acid was synthesized and its nitrosated products were shown to be mutagenic to Salmonella typhimurium TA 100 and TA 98. The potent mutagen Trp-P-2 is a typical 3-carboline derivative. The mutagenicity of Trp-P-2 was suppressed remarkably after nitrosation. Several 3-carboline derivatives also showed the similar property. Nitrosation of MTCA gave several derivatives which were isolated and showed direct mutagenicity to Salmonella typhimurium TA 98. Further characterization of these new carboline derivatives is in progress.     

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.     

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.     

Molecular epidemiology of cancer

In this review the role of molecular markers for the assessment of individual exposure to carcinogenic agents was analyzed. Examples of the studies describing mutation patterns related to specific carcinogenic exposures are presented. The results of epidemiological studies of gene polymorphism and its role in the interaction between inheritance, environmental factors, and lifestyles are analyzed in detail. Adequate planning and performance of the epidemiological component of a study is a requirement for obtaining reproducible results reflecting molecular mechanisms of interest. Individual information on lifestyle factors (smoking, alcohol consumption, nutrition, physical activity, reproductive anamnesis) and environmental factors (occupational activity and carcinogen load at workplace), which influence not only the risk of developing cancer, but also the molecular features of a tumor, is crucial for adequate analysis and proper assessment of the results.     

Quantum chemical studies of polycyclic aromatic hydrocarbons and their metabolites: correlations to carcinogenicity.

In the context of the bay region hypothesis for polycyclic aromatic hydrocarbon (PAH) carcinogenesis, molecular properties were calculated for seventeen polycyclic aromatic hydrocarbons related to (1) intrinsic substrate reactivities towards activating and detoxifying metabolism and (2) the stabilities of the putative carbocation ultimate carcinogens. All-valence electron methods were used, avoiding the inherent difficulties found in the pi-electron methods. The calculated substrate reactivities were found to predict major metabolites successfully, supporting the validity of their use in attempted correlations with observed carcinogenic potencies. Positive correlations were found between observed carcinogenic potencies and (1) the reactivities of the parent polycyclic aromatic hydrocarbons towards the initial distal bay region epoxidation and (2) the stabilities of the diol epoxide carbocations. The reactivities of the distal bay region diol epoxides, were high for both carcinogenic and non-carcinogenic compounds, implying that the second epoxidation does not determine relative carcinogenic activity. Support for a possible alternative hypothesis, that polycyclic aromatic hydrocarbons are activated by one electron oxidation, was also found.     

Metabolic activation capabilities of S9 and hepatocytes from uninduced rats to convert carcinogenic N-nitrosamines to mutagens

6 carcinogenic nitrosamines were studied in Salmonella typhimurium TA1535 after activation by S9 and by hepatocytes. All nitrosamines were activated by S9 from induced rats, regardless of their organotropy. The hepatocarcinogenic nitrosamines (N-nitrosodimethylamine, NDMA; N-nitrosodiethylamine, NDEA; N-nitrosomorpholine, NM and N-nitrosodibutylamine, NDBA) were activated to mutagens by S9 and by hepatocytes both derived from noninduced rat livers, NDMA and NM inducing more his+ revertants in the presence of hepatocytes. The oesophageal carcinogenic nitrosamine N-nitrosomethylbenzylamine (NMBeA) and bladder organotrophic N-nitroso(4-hydroxybutyl)butylamine(NBBOH) were neither converted by liver preparations of uninduced rats into mutagenic intermediates nor by hepatocytes. This study indicates that isolated cells derived from untreated animals may be better suited to study liver specific activation in vitro than disrupted subcellular metabolizing systems from induced animals.     

Inhibition ofin-vitro amino acid incorporation by the carcinogen N-methyl N’-nitro N-nitrosoguanidine

The addition of the carcinogen, N-methyl N’-nitro N-nitrosoguanidine, to a cell-free system consisting of purified polysome and ‘pH 5 enzyme’ fraction resulted in a marked inhibition of incorporation of (¹⁴C)-leucine into polypeptides. The extent of inhibition was remarkably high if the cell-free system contained limiting amount of ‘pH 5 enzyme’ fraction. Under this condition, the rate of inhibition was dependent on the concentration of carcinogen. Some component present in the ‘pH 5 enzyme’ fraction was inferred to be the susceptible factor, since the inhibition at low concentration of carcinogen could be reversed by increasing the amount of this fraction in the polysomal system. It was ascertained that tRNA was the primary target of carcinogenic action. Evidence suggested that functions attributed to tRNA such as aminoacylation and ribosomal transfer were both affected in a characteristic way by the action of the carcinogenic N-nitroso compound.     

Genotoxic and carcinogenic risk to humans of drug-nitrite interaction products

The large majority of N-nitroso compounds (NOC) have been found to produce genotoxic effects and to cause tumor development in laboratory animals; four NOC have been classified by the International Agency for Research on Cancer (IARC) as probably and another 15 as possibly carcinogenic to humans. A considerable fraction of drugs are theoretically nitrosatable due to the presence of amine, amide or other groups which by reacting with nitrite in the gastric environment, or even in other sites, can give rise to the formation of NOC, and in some cases other reactive species. This review provides a synthesis of information on the chemistry of NOC formation, the carcinogenic activity of NOC in animals and humans and the inhibitors of nitrosation reactions. It contains information on the drugs which have been tested for the formation of NOC by reaction with nitrite and the genotoxic-carcinogenic effects of their nitrosation products. In an extensive search we have found that 182 drugs, representing a wide variety of chemical structures and therapeutic activities, were examined in various experimental conditions for their ability to react with nitrite, and 173 (95%) of them were found to form NOC or other reactive species. Moreover, 136 drugs were examined in short-term genotoxicity tests and/or in long-term carcinogenesis assays, either in combination with nitrite or using their nitrosation product, in order to establish whether they produce genotoxic and carcinogenic effects; 112 (82.4%) of them have been found to give at least one positive response. The problem of endogenous drug nitrosation is largely unrecognized. Only a small fraction of theoretically nitrosatable drugs have been examined for the possible formation of genotoxic-carcinogenic NOC, guidelines for genotoxicity testing of pharmaceuticals do not indicate the need of performing the appropriate tests, and patients are not informed that the drug-nitrite interaction and the consequent risk can be reduced to a large extent by consuming the nitrosatable drug with ascorbic acid.     

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.     

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.     

A theoretical study of the carcinogenic nature of some aromatic amines

A hypothesis based on solubility, structural and electronic factors is used to predict the potential carcinogenic nature of 26 aromatic amines. The predictions are compared with the experimental data available on animals and humans and the agreement obtained is good. The results are useful in systematizing data available and in studying in detail aromatic amines whose carcinogenic activity is not definite.     

Various aspects of transplacental blastomogenesis in rabbits]

Transplacental blastomogenic action of different carcinogenic agents (ethylnitrosourea--ENU), methylnitrosourea--MNU, dimethylbenzanthracene--DMBA, and benz(a)pyrene--BP) was studied in rabbits. As revealed, ENU had the greatest blastomogenic activity and an expressed neurotropic action. Relative tropism to different tissues characteristic of some carcinogenic agents during their action on adult organism was not always expressed in transplacental blastomogenesis, Study of the influence of different modifying factors (organospecific immunization, chronic irritation of the peripheral nerve, injection of the MNU) on the realization of transplacental blastomogenesis showed that their postnatal use stimulated mostly the appearance of the peripheral nerve timours in the offspring rabbits (MNU inhibited the development of tumours).