An altered gene expression profile in tyramine-exposed intestinal cell cultures supports the genotoxicity of this biogenic amine at dietary concentrations.

Tyramine, histamine and putrescine are the most commonly detected and most abundant biogenic amines (BA) in food. The consumption of food with high concentrations of these BA is discouraged by the main food safety agencies, but legal limits have only been set for histamine. The present work reports a transcriptomic investigation of the oncogenic potential of the above-mentioned BA, as assessed in the HT29 human intestinal epithelial cell line. Tyramine had a greater effect on the expression of genes involved in tumorigenesis than did histamine or putrescine. Since some of the genes that showed altered expression in tyramine-exposed cells are involved in DNA damage and repair, the effect of this BA on the expression of other genes involved in the DNA damage response was investigated. The results suggest that tyramine might be genotoxic for intestinal cells at concentrations easily found in BA-rich food. Moreover, a role in promoting intestinal cancer cannot be excluded.

Interaction between the barley allelochemical compounds gramine and hordenine and artificial lipid bilayers mimicking the plant plasma membrane.

Some plants affect the development of neighbouring plants by releasing secondary metabolites into their environment. This phenomenon is known as allelopathy and is a potential tool for weed management within the framework of sustainable agriculture. While many studies have investigated the mode of action of various allelochemicals (molecules emitted by allelopathic plants), little attention has been paid to their initial contact with the plant plasma membrane (PPM). In this paper, this key step is explored for two alkaloids, gramine and hordenine, that are allelochemicals from barley. Using in vitro bioassays, we first showed that gramine has a greater toxicity than hordenine towards a weed commonly found in northern countries (Matricaria recutita L.). Then, isothermal titration calorimetry was used to show that these alkaloids spontaneously interact with lipid bilayers that mimic the PPM. The greater impact of gramine on the thermotropic behaviour of lipids compared to hordenine was established by means of infrared spectroscopy. Finally, the molecular mechanisms of these interactions were explored with molecular dynamics simulations. The good correlation between phytotoxicity and the ability to disturb lipid bilayers is discussed. In this study, biophysical tools were used for the first time to investigate the interactions of allelochemicals with artificial PPM.

The dietary biogenic amines tyramine and histamine show synergistic toxicity towards intestinal cells in culture.

Tyramine and histamine are the biogenic amines (BA) most commonly found at high concentrations in food; they may even appear together at toxic concentrations. The present work examines, via real-time cell analysis, whether histamine and tyramine show synergistic toxicity towards intestinal cell cultures. Employing a constant equipotency ratio, their interaction was examined via the combination index (CI) method of Chou & Talalay. Co-treatment with tyramine and histamine was associated with a stronger cytotoxic effect than was treatment with either BA or on its own. Indeed, a synergistic interaction (CI<1) was observed in the range of concentrations found in foods. The results also show that histamine, at concentrations below the legal limit, increases the cytotoxicity of tyramine at concentrations frequently reached in some foods. The synergistic cytotoxicity of tyramine and histamine should be taken into account when establishing legal limits designed to ensure consumer safety.

An analytical strategy to investigate Semen Strychni nephrotoxicity based on simultaneous HILIC-ESI-MS/MS detection of Semen Strychni alkaloids, tyrosine and tyramine in HEK 293t cell lysates.

A Previous metabolomics study has demonstrated that tyrosine metabolism might be disrupted by treating with Semen Strychni on the cell nephrotoxicity model. To investigate the relationship between Semen Strychni alkaloids (SAs) and endogenous tyrosine, tyramine under the nephrotoxicity condition, an HILIC-ESI-MS/MS based analytical strategy was applied in this study. Based on the established Semen Strychni nephrotoxicity cell model, strychnine and brucine were identified and screened as the main SAs by an HPLC-Q Exactive hybrid quadrupole Orbitrap mass system. Then, a sensitive HILIC-ESI-MS/MS method was developed to simultaneously monitor strychnine, brucine, tyrosine and tyramine in cell lysate. The analytes were separated by a Shiseido CAPCELL CORE PC (150mm×2.1mm, 2.7µm) HILIC column in an acetonitrile/0.1% formic acid gradient system. All the calibration curves were linear with regression coefficients above 0.9924. The absolute recoveries were more than 80.5% and the matrix effects were between 91.6%-107.0%. With the developed method, analytes were successfully determined in cell lysates. Decreased levels of tyrosine and tyramine were observed only in combination with increased levels of SAs, indicating that the disturbance of tyrosine metabolism might be induced by the accumulation of SAs in kidney cell after exposure of Semen Strychni. The HILIC-ESI-MS/MS based analytical strategy is a useful tool to reveal the relationships between the toxic herb components and the endogenous metabolite profiling in the toxicity investigation of herb medicines.

An Easily Fabricated Electrochemical Sensor Based on a Graphene-Modified Glassy Carbon Electrode for Determination of Octopamine and Tyramine.

A simple electrochemical sensor has been developed for highly sensitive detection of octopamine and tyramine by electrodepositing reduced graphene oxide (ERGO) nanosheets onto the surface of a glassy carbon electrode (GCE). The electrocatalytic oxidation of octopamine and tyramine is individually investigated at the surface of the ERGO modified glassy carbon electrode (ERGO/GCE) by using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). Several essential factors including the deposition cycle of reduced graphene oxide nanosheets and the pH of the running buffer were investigated in order to determine the optimum conditions. Furthermore, the sensor was applied to the quantification of octopamine and tyramine by DPV in the concentration ranges from 0.5 to 40 µM and 0.1 to 25 µM, respectively. In addition, the limits of detection of octopamine and tyramine were calculated to be 0.1 µM and 0.03 µM (S/N = 3), respectively. The sensor showed good reproducibility, selectivity and stability. Finally, the sensor successfully detected octopamine and tyramine in commercially available beer with satisfactory recovery ranges which were 98.5%-104.7% and 102.2%-103.1%, respectively. These results indicate the ERGO/GCE based sensor is suitable for the detection of octopamine and tyramine.

Comparative analysis of the in vitro cytotoxicity of the dietary biogenic amines tyramine and histamine.

Tyramine and histamine, the most toxic biogenic amines (BA), are often found in high concentrations in certain foods. Prompted by the limited knowledge of BA toxicity, and increasing awareness of the risks associated with high intakes of dietary BA, the in vitro cytotoxicity of tyramine and histamine was investigated. Tyramine and histamine were toxic for HT29 intestinal cell cultures at concentrations commonly found in BA-rich food, as determined by real-time cell analysis. Surprisingly, tyramine had a stronger and more rapid cytotoxic effect than histamine. Their mode of action was also different, while tyramine caused cell necrosis, histamine induced apoptosis. To avoid health risks, the BA content of foods should be reduced and legal limits established for tyramine.

N-Nicotinoyl tyramine, a novel niacinamide derivative, inhibits melanogenesis by suppressing MITF gene expression.

We synthesized and investigated the inhibitory effects of a novel niacinamide derivative, N-nicotinoyltyramine (NNT) on melanogenesis. NNT inhibited melanin production in B16F10 murine melanoma cells stimulated with α-melanocyte stimulating hormone (α-MSH), in human melanocyte and in three-dimensional cultured human skin model. NNT did not affect the catalytic activity of tyrosinase, but acted as an inhibitor of microphthalmia-associated transcription factor (MITF) and tyrosinase expressions in B16F10 cells. These findings suggest that the hypopigmentary effect of NNT results from the down-regulation of MITF and subsequently of tyrosinase, although NNT did not directly inhibit tyrosinase activity. In addition, safety of NNT was verified through performing neural stem cell morphology assay and Human repeated insult patch test as whitening agent. Our findings indicate that NNT may be a potential and non-skin irritant whitening agent for use in cosmetics and in the medical treatment of pigmentary disorders.

Enhanced toxicity to the catecholamine tyramine in polyglutamine transfected SH-SY5Y cells.

Huntington's disease (HD) is a progressive neurodegenerative disorder, of which the pathogenesis is not completely understood. In patients with Huntington's disease, there is a mutation in the gene encoding the protein huntingtin, which results in an expanded polyglutamine sequence leading to degeneration of the basal ganglia. There is mounting evidence that metabolism of the transmitter dopamine by the enzyme monoamine oxidase may contribute to striatal damage in mitochondrial toxin-induced models of HD. In this study, we have examined the role of the catecholamine tyramine in neural SH-SY5Y cells transfected with normal and expanded polyglutamine repeat numbers. Our findings demonstrate that cells containing a pathological number of polyglutamines are more sensitive to tyramine than cells with a non-pathological number. Tyramine-induced cell death was attenuated by MAO inhibitors as well as with catalase and the iron chelator deferoxamine, suggesting that H202 might mediate the observed toxicity. These observations support the notion that the metabolism of dopamine plays a role in neuron death in Huntington's disease.

Multiplex PCR for colony direct detection of Gram-positive histamine- and tyramine-producing bacteria.

Formation of biogenic amines (BA) may occur in fermented foods and beverages due to the amino acid decarboxylase activities of Gram-positive bacteria. These compounds may cause food poisoning and therefore could imply food exportation problems. A set of consensual primers based on histidine decarboxylase gene (hdc) sequences of different bacteria was designed for the detection of histamine-producing Gram-positive bacteria. A multiplex PCR based on these hdc primers and recently designed primers targeting the tyrosine decarboxylase (tyrdc) gene was created. A third set of primers targeting the 16S rRNA gene of eubacteria was also used as an internal control. This multiplex PCR was performed on extracted DNA as well as directly on cell colonies. The results obtained show that this new molecular tool allowed for the detection of Gram-positive histamine- and/or tyramine-producing bacteria. The use of this molecular tool for early and rapid detection of Gram-positive BA-producing bacteria is of interest in evaluating the potential of cultured indigenous strains to produce biogenic amines in a fermented food product as well as to validate the innocuity of potential starter strains in the food industry.

Tyramine functions as a toxin in honey bee larvae during Varroa-transmitted infection by Melissococcus pluton.

From wounds of honey bee pupae, caused by the mite Varroa destructor, coccoid bacteria were isolated and identified as Melissococcus pluton. The bacterial isolate was grown anaerobically in sorbitol medium to produce a toxic compound that was purified on XAD columns, gelfiltration and preparative HPLC. The toxic agent was identified by GC-MS and FTICR-MS as tyramine. The toxicity of the isolated tyramine was tested by a novel mobility test using the protozoon Stylonychia lemnae. A concentration of 0.2 mg/ml led to immediate inhibition of mobility. In addition the toxicity was studied on honey bee larvae by feeding tyramine/water mixtures added to the larval jelly. The lethal dosis of tyramine on 4-5 days old bee larvae was determined as 0.3 mg/larvae when added as a volume of 20 microl to the larval food in brood cells. Several other biogenic amines, such as phenylethylamine, histamine, spermine, cadaverine, putrescine and trimethylamine, were tested as their hydrochloric salts for comparison and were found to be inhibitory in the Stylonychia mobility test at similar concentrations. A quantitative hemolysis test with human red blood cells revealed that tyramine and histamine showed the highest membranolytic activity, followed by the phenylethylamine, trimethylamine and spermine, while the linear diamines, cadaverine and putrescine, showed a significantly lower hemolysis when calculated on a molar amine basis. The results indicate that tyramine which is a characteristic amine produced by M. pluton in culture, is the causative agent of the observed toxic symptoms in bee larvae. Thus this disease, known as European foulbrood, is possibly an infection transmitted by the Varroa destructor mite.

Tumor promotion of N-nitroso-N-(3-keto-1, 2-butanediol)-3'-nitrotyramine derived from nitrosation of Maillard reaction product in CD-1 mice.

N-Nitroso-N-(3-keto-1,2-butanediol)-3'-nitrotyramine (NO-NTA) is a product of a model browning system generated in the presence of sodium nitrite. Our previous study showed that NO-NTA had genotoxicity and proved to be an initiator and promoter on mouse C3H10T1/2 cells. In this study, a two-stage skin carcinogenesis protocol was used to promote CD-1 mouse skin carcinogenesis using NO-NTA. Twice weekly, for 38 weeks, topical application of NO-NTA at the concentration of 250 nmol to mice previously initiated with benzo(a)pyrene (BaP) caused 90% tumor incidence. However, no tumors were observed in mice treated with BaP or treated with NO-NTA alone. The NO-NTA-promoted tumors that were observed histologically in mice showed well-differentiated squamous cell carcinoma with invasion into the subcutaneous region. Application of the same amount of NO-NTA not only caused significant induction of hyperplasia but also epidermal ornithine decarboxylase (ODC) activity. Treatment of mouse skin (1 cm(2)) with various amounts of NO-NTA (10, 50, or 250 nmol) caused production of hydrogen peroxide by 1.63-, 1.91-, and 2. 38-fold, respectively, and marked induction of myeloperoxidase (MPO) by 21-, 39-, and 61-fold. These results indicate that NO-NTA is a new tumor promoter and may induce tumor promotion by oxidant stress in CD-1 mouse skin.

Tumor promoting effect of N-nitroso-N-(2-hexanonyl)-3'-nitrotyramine (a nitrosated Maillard reaction product) in benzo(a)pyrene-initiated mouse skin carcinogenesis.

N-nitroso-N-(2-hexanonyl)-3'-nitrotyramine (NO-HNTA) is a product generated in a model browning system in the presence of sodium nitrite. The chemical structure of this compound has been confirmed by UV, mass, nuclear magnetic resonance and infrared spectroscopy in our study. Twenty weeks, twice weekly, topical application of NO-HNTA at the concentration of 10, 50 and 250 mumol to mice previously initiated with benzo(a)pyrene (B[a]P) increased their tumor formation by 3.2-, 4.6- and 5.8-fold respectively. Application of the same amount of NO-HNTA not only caused significant induction of hyperplasia but also the activity of epidermal ornithine decarboxylase (ODC). Treatment of mouse skin with various amounts of NO-HNTA (10, 50 and 250 mumol) caused production of hydrogen peroxide by 1.38-, 1.95- and 3.26-fold respectively, and induction myeloperoxidase (MPO) by 24-, 63- and 102-fold. These results indicate that the formation of NO-HNTA or its derivatives derived from the reaction of tyrosine and glucose in the presence of sodium nitrite has the potential as a tumor promoter.

Inhibitory effects of citrus fruits on the mutagenicity of 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid treated with nitrite in the presence of ethanol.

It has been shown that the mutagenicity of 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid (MTCCA), a mutagen precursor in soy sauce treated with nitrite, was strongly increased when it was treated with nitrite in the presence of alcohols. We found that the mutagenicity of MTCCA treated with 50 mM nitrite at pH 3, 37 degrees C for 60 min in the presence of 7.5% ethanol was reduced by the addition of citrus fruits sudachi (Citrus sudachi), lemon (C. limon) and yuzu (C. junos), to the reaction mixture. The mutagenicity-reducing activity per weight of flavedos (outer colored portions of peel) of the citrus fruits was considerably higher than that of the juices. The juices of the other citrus fruits commercially available in Japan also had mutagenicity-reducing activity against the nitrite-treated MTCCA. Among the many components of citrus fruits, dietary fibers lignin and pectin showed strong antimutagenic activity in the reaction mixture, suggesting that the mixed fractions of these components including lignin, pectin, D-limonene, naringin, hesperidin, ascorbic acid and citric acid reduce the mutagenicity of MTCCA in the reaction mixture containing nitrite and ethanol.

Toxicity of Palicourea marcgravii: combined effects of fluoroacetate, N-methyltyramine and 2-methyltetrahydro-beta-carboline.

Feeding experiments carried out with cattle and horses could prove the toxic effects of P. marcgravii (Rubiaceae) in all cases. The typical symptoms of "sudden death", however, are observed in ruminants only. This difference could not be explained so far. Apart from fluoroacetate, two more substances also have influence the toxic effects and have been isolated from P. marcgravii for the first time: N-methyltyramine and 2-methyltetrahydro-beta-carboline (2-Me THBC). Structure elucidation of these compounds is mainly accomplished by 1H-NMR, 13C-NMR and MS techniques. Due to the small quantity of fluoroacetate (5.4 micrograms/g plant), the main toxic effect obviously lies in the two discovered substances. In contrast to the slow death of horses (monogastriers), the "sudden death syndrome" of cattle (ruminants) can be explained as a result of the higher resorbility of these two substances in the gastro-intestinal system. Given orally, both substances influence the monoamine oxidase type A (MAO-A): N-methyltyramine acts as a competitive substrate, and 2-Me THBC is one of the most effective MAO-A-inhibitors. Thus, the decomposition of the specific MAO-A-substrates noradrenaline and adrenaline as well as of N-methyltyramine itself is inhibited. The alpha- and beta-receptors of the sympathetic system are stimulated more strongly, which leads to a drastic rise in blood pressure and thereby to a more rapid distribution of fluoroacetate in the body. This results in a reinforced input of fluoroacetate in the cells of especially active organs of the body (heart etc.). Thus, even smaller quantities of fluoroacetate are lethal.

Mutagenicity of soy sauce treated with nitrite in the presence of ethanol or alcoholic beverages.

The mutagenicity induced by soy sauce after reaction with 50 mM nitrite at pH 3, 37 degrees C, for 60 min in the presence of 1.25-10% ethanol was reduced in proportion to the ethanol concentration. The mutagenicity of soy sauce treated with nitrite was also reduced in the presence of commercial alcoholic beverages, Japanese sake, wine, 'shochu', whiskey and brandy, but not beer, in proportion to the concentration. The mutagenicity of nitrite-treated tyramine, which is a major precursor of a mutagen in soy sauce treated with nitrite, was strongly reduced in the presence of ethanol, n-propanol or isopropanol and more strongly reduced in the presence of methanol, but was increased twofold in the presence of the sugars glucose or sucrose. The reduction of the mutagenicity of nitrite-treated tyramine required simultaneous treatment of tyramine with ethanol and nitrite. The mutagenicity of tyramine treated with nitrite was clearly reduced in the presence of shochu and whiskey, similarly to ethanol. Analysis by high-performance liquid chromatography revealed that the reduction of the mutagenicity of nitrite-treated tyramine in the presence of ethanol resulted from the reduced production of mutagenic 3-diazotyramine from tyramine.

[Mutagenicity of tyramine extracted from salted dried fish in high-risk area of gastric cancer in Zhuanghe county].

Content of tyramine was determined in salted and dried small fish collected from high-risk area of gastric cancer in Zhuanghe County, Liaoning Province with high pressure liquid chromatography, and its mutagenicity after nitrosification was assayed. Results showed content of tyramine in the fish correlated significantly with its mutagenicity (r = 0.993, and P < 0.01). And, tyramine extracted from the fish was verified by thin layer chromatography.

Mutagenic activity of dopamine after nitrosation.

Dopamine hydrochloride is reported to be a new mutagen precursor in this study. After treatment with nitrite under acidic conditions, dopamine hydrochloride showed direct-acting mutagenicity on Salmonella typhimurium TA100, TA98 and Escherichia coli WP2uvra. The addition of S9 mix did not affect the mutagenicity of nitrosated dopamine significantly in these three strains. Meanwhile, a comparison of the mutagenicity of nitrosated dopamine with nitrosated tyramine was carried out.

N-Nitroso-N-(3-keto-1,2-butanediol)-3'-nitrotyramine. A new genotoxic agent derived from the reaction of tyrosine and glucose in the presence of sodium nitrite.

N-Nitroso-N-(3-keto-1,2-butanediol)-3'-nitrotyramine (NO-NTA) is a product of a model browning system in the presence of sodium nitrite. In this study, the chemical structure is confirmed by spectral studies, including UV, mass spectrometry, nuclear magnetic resonance and infrared spectroscopy. NO-NTA is strongly genotoxic to the rat hepatocyte and is moderately cytotoxic to mouse C3H10T1/2 cells. Results obtained in this study indicate that NO-NTA inflicted DNA damage through the formation of a DNA adduct. In addition, C3H10T1/2 cells were treated with NO-NTA and, following addition of 12-O-tetradecanoylphorbol-13-acetate (TPA) as promotor, the increase of transformed foci indicated that NO-NTA could possibly be an initiator [corrected] of TPA tumor promotion. A transformed cell line from NO-NTA initiated and TPA promoted foci increased saturation density and growth ability in soft agar reactive to the control line. These results suggest that the formation of a genotoxic agent of nitroso-derivatives may take place in a nitrite-containing food system during processing and cooking.

Genotoxicity of nitrosated red wine and of the nitrosatable phenolic compounds present in wine: tyramine, quercetin and malvidine-3-glucoside.

Phenolic compounds and biogenic amines are known to be present in some foodstuffs which become directly genotoxic after nitrosation in vitro. Red wine has previously been shown to be genotoxic and this activity has been attributed mainly to flavonoids. Besides flavonoids, red wine contains a multiplicity of compounds, including biogenic amines. Using the Ames assay and the SOS chromotest, this study has shown that red wine and some of the nitrosatable molecules present in wine become directly genotoxic on nitrosation in vitro: these include the phenolic molecules tyramine, quercetin and malvidine-3-glucoside, whereas phenylethylamine and histamine were negative on nitrosation. Interestingly, quercetin had been predicted to be negative after nitrosation, using the CASE methodology. The concentrations of these three positive nitrosatable compounds in wine were determined by HPLC. Comparison of these concentrations and their respective levels of genotoxicity suggests that the genotoxicity after nitrosation is probably attributable to other molecules. It is also possible that synergistic effects may occur between various nitrosatable compounds in wine.