Biosynthesis of vitamin B3 and NAD+: incubating HepG2 cells with the alkaloid myosmine.

BACKGROUND: In the kynurenine pathway, it is reported that the essential amino acid tryptophan forms nicotinic acid (NA, vitamin B3) in biological systems. This pathway is part of the de novo pathway to perform nicotinamide adenine dinucleotide (NAD+) biosynthesis. Additionally, biosynthesis of NAD+ via the Preiss-Handler pathway involves NA and its analogue nicotinamide, both designated as niacin. Previous attempts were successful in converting myosmine (MYO) by organic synthesis to NA, and the assumption was that the alkaloid MYO, which is taken in from food, can be converted into NA by biological oxidation. RESULT: Incubation of HepG2 cells with MYO yielded NA. Moreover, a significant increase of NAD+ compared with the control has been found. CONCLUSION: Hence, MYO could be assumed to be the hitherto unknown origin of an alternative NA biosynthesis additionally influencing NAD+ biosynthesis positively. This novel MYO pathway may open new perspectives to improve knowledge and relevance of NA and NAD+ biosynthesis and bioactivation in cells and, moreover, in food staples, food, and diet. © 2024 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Classical transient receptor potential 6 (TRPC6) channels support myofibroblast differentiation and development of experimental pulmonary fibrosis.

Pulmonary fibrosis (PF) is a chronic progressive lung disease without effective medical treatment options leading to respiratory failure and death within 3-5years of diagnosis. The pathological process of PF is driven by aberrant wound-healing involving fibroblasts and myofibroblasts differentiated by secreted profibrotic transforming growth factor ß (TGF-ß1). Classical transient receptor potential 6 (TRPC6), a Na+- and Ca2+-permeable cation channel, is able to promote myofibroblast conversion of primary rat cardiac and human dermal fibroblasts and TRPC6-deficiency impaired wound healing after injury. To study a potential role of TRPC6 in the development of PF we analyzed lung function, gene and protein expression in wild-type (WT) and TRPC6-deficient (TRPC6-/-) lungs utilizing a bleomycin-induced PF-model. Fibrotic WT-mice showed a significant higher death rate while bleomycin-treated TRPC6-deficient mice were partly protected from fibrosis as a consequence of a lower production of collagen and an almost normal function of the respiratory system (reduced resistance and elastance compared to fibrotic WT-mice). On a molecular level TGF-ß1 induced TRPC6 up-regulation, increased Ca2+ influx and nuclear NFAT localization in WT primary murine lung fibroblasts (PMLFs) resulting in higher stress fiber formation and accelerated contraction rates as compared to treated TRPC6-deficient fibroblasts. Therefore, we conclude that TRPC6 is an important determinant for TGF-ß1-induced myofibroblast differentiation during fibrosis and specific channel inhibitors might be beneficial in a future treatment of PF.

Isolation of Nicotinic Acid (Vitamin B3) and N-Propylamine after Myosmine Peroxidation.

The alkaloid myosmine (3-(1-pyrroline-2-yl)pyridine) is widespread in biological matrixes including foodstuffs and tobacco products. Some in vitro tests in cellular systems showed mutagenic activity for myosmine. Myosmine activation including peroxidation mechanism employs unstable oxazirane intermediates. The formation of minor metabolite 3-hydroxymethyl-pyridine in rat metabolism experiments as well as in in vitro peroxidation assays suggests its further oxidation to nicotinic acid and possible concomitant formation of n-propylamine. A sensitive high-performance liquid chromatography-ultraviolet (HPLC-UV) method was developed for the direct analysis of n-propylamine in the peroxidation assay solution of myosmine employing derivatization with 3,5-dinitrobenzoyl chloride. Additionally, during peroxidation procedures, formation of 3-pyridylmethanol to nicotinic acid, the essential vitamin B3, was observed and characterized using HPLC-UV and gas chromatography/mass spectrometry. This new reaction pathway may present further contribution to our knowledge of myosmine's significance in human food including its activation in human organism, foodstuffs, and biological systems.

Tissue distribution and excretion of myosmine after i.v. administration to Long-Evans rats using quantitative whole-body autoradiography.

Occurrence of the tobacco alkaloid myosmine has been proven in various staple foods, vegetables and fruits. Myosmine can be easily activated by nitrosation yielding 4-hydroxy-1-(3-pyridyl)-butanone (HPB) and the esophageal carcinogen N'-nitrosonornicotine. Most of the reaction products after myosmine peroxidation were also identified as urinary metabolites after oral administration to rats. Whole-body autoradiography with freeze dried or multiple solvent extracted tissue sections was used to trace [2'-(14)C]myosmine (0.1 mCi/kg bw) 0.1, 0.25, 1, 4 and 24 h after i.v. injection in Long-Evans rats. In addition, in vitro binding of radioactivity to esophageal and eye tissue was determined and excretion of radioactivity via urine and feces was quantified. Radioactivity is rapidly eliminated by renal excretion. Approximately 30% of the administered radioactivity was recovered in urine within the first 4 h and excretion with urine (72%) and feces (15%) was nearly complete after 24 h. A rapid concentration of radioactivity can be seen in the stomach and in the salivary and lachrymal glands. Rats killed 1 and 4 h after treatment showed by far the highest labeling in the accessory genital gland. High levels of nonextractable radioactivity were present in esophageal tissue and melanin. The half lives for the disappearance of radioactivity from various tissues are in the order of about 1 h. Eye and esophagus sections both showed nonextractable labeling after in vitro incubation with (14)C-myosmine. In conclusion, the toxicological significance of myosmine accumulation in esophagus and accessory genital gland requires further investigations. Hair analysis might be applicable for myosmine biomonitoring, because of possible enrichment in melanin containing tissues.

Reaction of the tobacco alkaloid myosmine with hydrogen peroxide.

Myosmine is not only one of the minor tobacco alkaloids but is also present in various foods. Therefore, research on myosmine metabolism and activation has been intensified. 3-Pyridylacetic acid, 4-oxo-4-(3-pyridyl)butanoic acid (keto acid), 3-pyridylmethanol, 3'-hydroxymyosmine, and 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) have been identified as urinary metabolites after oral administration to female Wistar rats. Although N-nitrosation of myosmine, yielding N'-nitrosonornicotine (NNN) and HPB, was considered as a possible in vivo activation route, the formation pathways of most metabolites could not be explained until now. Therefore, under consideration of its high reactivity due to its imine structure, peroxidation of myosmine seemed to be a promising additional activation pathway. In vitro peroxidation using myosmine (8.9 micromol in 200 microL methanol) with a mixture of hydrogen peroxide (57.6 micromol, 5 microL of a 35% solution) and acetic acid anhydride (106 micromol, 10 microL) already showed high yields of reaction products after 30 min ultrasonic treatment. The product pattern was analyzed by HPLC/UV and GC/MS. Besides unchanged myosmine, 3-pyridylacetic acid, keto acid, 3-pyridylmethanol, HPB, and nornicotyrine have been identified as myosmine peroxidation products. Different product patterns were obtained after 24 h and 4 days due to a time-dependent degradation, formation, and conversion of the reaction products. Therefore, peroxidation reaction of myosmine might explain the in vivo formation of 3-pyridylacetic acid, keto acid, 3-pyridylmethanol, and HPB in rats. In addition, because of acetylating conditions using acetic acid anhydride, N-(4-oxo-4-pyridin-3-yl-butyl)acetamide was rapidly formed during the first 30 min of the reaction.

Metabolism of myosmine in Wistar rats.

The alkaloid myosmine is present not only in tobacco products but also in various foods. Myosmine is easily nitrosated, yielding 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) and the esophageal tobacco carcinogen N'-nitrosonornicotine. Due to its widespread occurrence, investigations on the metabolism and activation of myosmine are needed for risk assessment. Therefore, the metabolism of myosmine has been studied in Wistar rats treated with single oral doses of [pyridine-5-3H]myosmine at 0.001, 0.005, 0.5, and 50 micromol/kg body weight. Oral administration was achieved by feeding a labeled apple bite. Radioactivity was completely recovered in urine and feces within 48 h. At the two lower doses, 0.001 and 0.005 micromol/kg, a higher percentage of the radioactivity was excreted in urine (86.2 +/- 4.9% and 88.9 +/- 1.7%) as compared with the higher doses, 0.5 and 50 micromol/kg, where only 77.8 +/- 7.3% and 75.4 +/- 6.6% of the dose was found in urine. Within 24 h, urinary excretion of radioactivity was nearly complete with less than 4% of the total urinary output appearing between 24 and 48 h. The two major metabolites accounting for >70% of total radioactivity in urine were identified as 3-pyridylacetic acid (20-26%) and 4-oxo-4-(3-pyridyl)butyric acid (keto acid, 50-63%) using UV-diode array detection and gas chromatography-mass spectrometry measurements. 3-Pyridylmethanol (3-5%), 3'-hydroxymyosmine (2%) and HPB (1-3%) were detected as minor metabolites. 3'-Hydroxymyosmine is exclusively formed from myosmine and therefore might be used as a urinary biomarker for myosmine exposure in the future.

Acute and subacute effects of tobacco alkaloids, tobacco-specific nitrosamines and phenethyl isothiocyanate on N'-nitrosonornicotine metabolism in rats.

N'-Nitrosonornicotine (NNN) was the first tobacco-specific nitrosamine (TSNA) identified as carcinogen in tobacco smoke, but no data exist on in vivo interactions between NNN and other tobacco alkaloids, TSNA or phenethyl isothiocyanate (PEITC) which have been demonstrated in various studies on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Acute effects on NNN metabolism were tested in male Fischer F344 rats injected s.c. with 30nmol/kg body weight (bw) [5-(3)H]NNN either alone or simultaneously with 15mumol/kg bw nicotine, nornicotine, anatabine, or anabasine, 150mumol/kg bw cotinine, 3mumol/kg bw myosmine, or 300nmol/kg bw of either N'-nitrosoanatabine or N'-nitrosoanabasine. Another group of rats was fed a diet supplemented with PEITC at 1mumol/g diet starting 24h before NNN treatment. Within 24h more than 80% and about 10% of the radioactivity was excreted with urine and feces, respectively. Urinary metabolites were separated by reversed-phase radio-HPLC and identified by co-chromatography with UV standards. In two sets of experiments with control rats treated with NNN only, 4-hydroxy-4-(3-pyridyl)butanoic acid (hydroxy acid, 44.4/44.8%), 4-oxo-4-(3-pyridyl)butanoic acid (keto acid, 32.4/31.5%), NNN-N-oxide (5.0/3.8%), 4-(3-pyridyl)butane-1,4-diol (diol, 1.1/1.0%) and norcotinine (2.3/1.0%) were consistently detected besides unmetabolised NNN (4.7/3.3%). Co-treatment with nicotine, cotinine, nornicotine and PEITC shifted the contribution of the two major metabolites significantly in favor of hydroxy acid (108-113% of control) as compared to keto acid (86-90% of control). The same treatments also increased norcotinine (135-170% of control). These changes are consistent with a decreased metabolic activation of NNN. In subacute studies rats received NNN in drinking water for 4 weeks at a daily dose of 30 nmol/kg bw with or without nornicotine at 15 micromol/kg bw or myosmine at 3 micromol/kg bw. On the last day of the experiment all rats received [5-(3)H]NNN at 30 nmol/kg bw with a contaminated apple bite followed by collection of urine and feces for 18h. Most of the radioactivity, 87-96% of the dose, was recovered in urine and only minor amounts have been excreted in feces or persisted in blood. In urine of the NNN-control group keto acid (32.2%) and unmetabolised NNN (3.9%) were present in identical amounts as in the acute experiment whereas hydroxy acid (41.4% of total radioactivity in urine, 93% of acute NNN control) was reduced in expense of the minor NNN metabolites. Co-administration of nornicotine resulted in a small but significant rise of keto acid (107% of control) and a significant decrease in NNN-N-oxide (76% of control). After co-treatment with myosmine the increase of keto acid (104% of control) was even less but still significant whereas NNN-N-oxide and diol were significantly reduced to 72% and 79% of control, respectively. Our experiments with rats indicate significant mutual effects of some of the major tobacco alkaloids and most relevant TSNA. Further studies on the impact on smokers and the inhibitory effects of isothiocyanates are needed for a final risk assessment.

Genotoxic effects of myosmine in human lymphocytes and upper aerodigestive tract epithelial cells.

Myosmine, 3-(1-pyrroline-2-yl)pyridine, is an alkaloid found in tobacco plants. Recently, it was also detected in various edibles and staple foods. Whereas other tobacco alkaloids such as nicotine and nornicotine and their nitrosation products, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN), have been widely discussed, the mutagenic impact of myosmine has not been investigated in detail. In the present study, possible genotoxic effects of myosmine were studied in human lymphocytes and nasal mucosal cells using the alkaline single cell microgel electrophoresis (Comet) assay. DNA single strand breaks, alkali labile sites and incomplete excision repair sites were expressed using the Olive tail moment (OTM). One hour incubation with myosmine at 0, 5, 10, 25 and 50 mM induced a low but significantly dose-dependent increase of DNA migration from 1.29 +/- 0.13 to 18.25 +/- 1.59 (OTM, mean +/- S.E., N=11) in lymphocytes. In nasal mucosal cells a similar although somewhat less extensive DNA damage from 1.17 +/- 0.12 to 21.67 +/- 2.97 (OTM, mean +/- S.E., N=10-11) was obtained after 1 h incubation with myosmine at 0, 10, 25, 50 and 100 mM. After prolonged incubation of human lymphocytes with 10mM myosmine for 1, 3, 6, and 24 h, a significant time-dependent increase of DNA migration from 3.45 +/- 0.43 to 57.77 +/- 8.24 (OTM, mean +/- S.E., N=4) was observed. Our data indicate that myosmine expresses significant genotoxic effects in human target cells of carcinogenesis. This result warrants further investigations on the impact of this dietary component on human health.

New sources of dietary myosmine uptake from cereals, fruits, vegetables, and milk.

Myosmine has been regarded as a specific tobacco alkaloid until investigations pointed out that nuts and nut products constitute a significant source of myosmine. In the present study it is shown that the occurrence of myosmine is widespread throughout a large number of plant families. Using a method for extraction practicable for all examined foods, quantitative analysis through internal standard addition showed nanograms per gram amounts. Positively tested edibles were staple foods such as maize, rice, wheat flour, millet, potato, and milk and also cocoa, popcorn, tomato, carrot, pineapple, kiwi, and apples. No myosmine was detectable in other vegetables and fruits such as lettuce, spinach, cucumber, onion, banana, tangerines, and grapes. Myosmine is easily nitrosated giving rise to a DNA adduct identical to the esophageal tobacco carcinogen N-nitrosonornicotine. Therefore, the role of dietary myosmine in esophageal adenocarcinoma should be further investigated.