The Impact of NAD Bioavailability on DNA Double-Strand Break Repair Capacity in Human Dermal Fibroblasts after Ionizing Radiation.

Nicotinamide adenine dinucleotide (NAD) serves as a substrate for protein deacetylases sirtuins and poly(ADP-ribose) polymerases, which are involved in the regulation of DNA double-strand break (DSB) repair molecular machinery by various mechanisms. However, the impact of NAD bioavailability on DSB repair remains poorly characterized. Herein, using immunocytochemical analysis of γH2AX, a marker for DSB, we investigated the effect of the pharmacological modulation of NAD levels on DSB repair capacity in human dermal fibroblasts exposed to moderate doses of ionizing radiation (IR). We demonstrated that NAD boosting with nicotinamide riboside did not affect the efficiency of DSB elimination after the exposure of cells to IR at 1 Gy. Moreover, even after irradiation at 5 Gy, we did not observe any decrease in intracellular NAD content. We also showed that, when the NAD pool was almost completely depleted by inhibition of its biosynthesis from nicotinamide, cells were still able to eliminate IR-induced DSB, though the activation of ATM kinase, its colocalization with γH2AX and DSB repair capacity were reduced in comparison to cells with normal NAD levels. Our results suggest that NAD-dependent processes, such as protein deacetylation and ADP-ribosylation, are important but not indispensable for DSB repair induced by moderate doses of IR.

Two-Photon Excited Fluorescence of NADH-Alcohol Dehydrogenase Complex in a Mixture with Bacterial Enzymes.

Thorough study of composition and fluorescence properties of a commercial reagent of active equine NAD-dependent alcohol dehydrogenase expressed and purified from E. coli has been carried out. Several experimental methods: spectral- and time-resolved two-photon excited fluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, fast protein liquid chromatography, and mass spectrometry were used for analysis. The reagent under study was found to contain also a number of natural fluorophores: free NAD(P)H, NADH-alcohol dehydrogenase, NADPH-isocitrate dehydrogenase, and pyridoxal 5-phosphate-serine hydroxymethyltransferase complexes. The results obtained demonstrated the potential and limitations of popular optical methods as FLIM for separation of fluorescence signals from free and protein-bound forms of NADH, NADPH, and FAD that are essential coenzymes in redox reactions in all living cells. In particular, NADH-alcohol dehydrogenase and NADPH-isocitrate dehydrogenase complexes could not be optically separated in our experimental conditions although fast protein liquid chromatography and mass spectrometry analysis undoubtedly indicated the presence of both enzymes in the molecular sample used. Also, the results of fluorescence, fast protein liquid chromatography, and mass spectrometry analysis revealed a significant contribution of the enzyme-bound coenzyme pyridoxal 5-phosphate to the fluorescence signal that could be separated from enzyme-bound NADH by using bandpass filters, but could effectively mask contribution from enzyme-bound FAD because the fluorescence spectra of the species practically overlapped. It was shown that enzyme-bound pyridoxal 5-phosphate fluorescence can be separated from enzyme-bound NAD(P)H and FAD through analysis of short fluorescence decay times of about tens of picoseconds. However, this analysis was found to be effective only at relatively high number of peak photon counts in recorded fluorescence signals. The results obtained in this study can be used for interpretation of fluorescence signals from a mixture of enzyme-bound fluorophores and should be taken into consideration when determining the intracellular NADH/FAD ratio using FLIM.

Purine nucleoside phosphorylase controls nicotinamide riboside metabolism in mammalian cells.

Nicotinamide riboside (NR) is an effective precursor of nicotinamide adenine dinucleotide (NAD) in human and animal cells. NR supplementation can increase the level of NAD in various tissues and thereby improve physiological functions that are weakened or lost in experimental models of aging or various human pathologies. However, there are also reports questioning the efficacy of NR supplementation. Indeed, the mechanisms of its utilization by cells are not fully understood. Herein, we investigated the role of purine nucleoside phosphorylase (PNP) in NR metabolism in mammalian cells. Using both PNP overexpression and genetic knockout, we show that after being imported into cells by members of the equilibrative nucleoside transporter family, NR is predominantly metabolized by PNP, resulting in nicotinamide (Nam) accumulation. Intracellular cleavage of NR to Nam is prevented by the potent PNP inhibitor Immucillin H in various types of mammalian cells. In turn, suppression of PNP activity potentiates NAD synthesis from NR. Combining pharmacological inhibition of PNP with NR supplementation in mice, we demonstrate that the cleavage of the riboside to Nam is strongly diminished, maintaining high levels of NR in blood, kidney, and liver. Moreover, we show that PNP inhibition stimulates Nam mononucleotide and NAD+ synthesis from NR in vivo, in particular, in the kidney. Thus, we establish PNP as a major regulator of NR metabolism in mammals and provide evidence that the health benefits of NR supplementation could be greatly enhanced by concomitant downregulation of PNP activity.

Welcome to the Family: Identification of the NAD+ Transporter of Animal Mitochondria as Member of the Solute Carrier Family SLC25.

Subcellular compartmentation is a fundamental property of eukaryotic cells. Communication and metabolic and regulatory interconnectivity between organelles require that solutes can be transported across their surrounding membranes. Indeed, in mammals, there are hundreds of genes encoding solute carriers (SLCs) which mediate the selective transport of molecules such as nucleotides, amino acids, and sugars across biological membranes. Research over many years has identified the localization and preferred substrates of a large variety of SLCs. Of particular interest has been the SLC25 family, which includes carriers embedded in the inner membrane of mitochondria to secure the supply of these organelles with major metabolic intermediates and coenzymes. The substrate specificity of many of these carriers has been established in the past. However, the route by which animal mitochondria are supplied with NAD+ had long remained obscure. Only just recently, the existence of a human mitochondrial NAD+ carrier was firmly established. With the realization that SLC25A51 (or MCART1) represents the major mitochondrial NAD+ carrier in mammals, a long-standing mystery in NAD+ biology has been resolved. Here, we summarize the functional importance and structural features of this carrier as well as the key observations leading to its discovery.

Safety Assessment of Bacillus subtilis MB40 for Use in Foods and Dietary Supplements.

With the growing popularity of probiotics in dietary supplements, foods, and beverages, it is important to substantiate not only the health benefits and efficacy of unique strains but also safety. In the interest of consumer safety and product transparency, strain identification should include whole-genome sequencing and safety assessment should include genotypic and phenotypic studies. Bacillus subtilis MB40, a unique strain marketed for use in dietary supplements, and food and beverage, was assessed for safety and tolerability across in silico, in vitro, and in vivo studies. MB40 was assessed for the absence of undesirable genetic elements encoding toxins and mobile antibiotic resistance. Tolerability was assessed in both rats and healthy human volunteers. In silico and in vitro testing confirmed the absence of enterotoxin and mobile antibiotic resistance genes of safety concern to humans. In rats, the no-observed-adverse-effect level (NOAEL) for MB40 after repeated oral administration for 14 days was determined to be 2000 mg/kg bw/day (equivalent to 3.7 × 1011 CFU/kg bw/day). In a 28 day human tolerability trial, 10 × 109 CFU/day of MB40 was well tolerated. Based on genome sequencing, strain characterization, screening for undesirable attributes and evidence of safety by appropriately designed safety evaluation studies in rats and humans, Bacillus subtilis MB40 does not pose any human health concerns under the conditions tested.

Genetic Toxicity Studies of the Ketogenic Ester Bis Hexanoyl (R)-1,3-Butanediol.

A series of studies was conducted to assess the genetic toxicity of a novel ketone ester, bis hexanoyl (R)-1,3-butanediol (herein referred to as BH-BD), according to Organization for Economic Co-operation and Development testing guidelines under the standards of Good Laboratory Practices. In bacterial reverse mutation tests, there was no evidence of mutagenic activity in any of the Salmonella typhimurium strains tested or in Escherichia coli strain WP2uvrA, at dose levels up to 5,000 µg/plate in the presence or absence of Aroclor 1254-induced rat liver (S9 mix) for metabolic activation. In the in vitro micronucleus test using human TK6 cells, BH-BD did not show a statistically significant increase in the number of cells containing micronuclei when compared with concurrent control cultures at all time points and at any of the concentrations analyzed (up to 100 µg/mL, final concentration in culture medium), with and without S9 mix activation. In the in vivo micronucleus test using Sprague Dawley rats, BH-BD did not show a statistically significant increase in the incidence of micronucleated polychromatic erythrocytes relative to the vehicle control group. Therefore, BH-BD was concluded to be negative in all 3 tests. These results support the safety assessment of BH-BD for potential use in food.

Toxicological evaluation of the ketogenic ester bis hexanoyl (R)-1,3-butanediol: Subchronic toxicity in Sprague Dawley rats.

Bis-hexanoyl (R)-1,3-butanediol (BH-BD) is novel ketone ester undergoing development as a food ingredient to achieve nutritional ketosis in humans. Male and female Crl:CD(SD) rats were administered BH-BD twice daily at 9000, 12,000 or 15,000 mg/kg/day, by oral gavage in a 90-day toxicity study with 28-day recovery period; and an interim 28-day phase. Test substance-related early deaths occurred in four females at 15,000 mg/kg/day. A dose-dependent increase in acute transient postdose (1-3 h) observations of incoordination at ≥12,000 mg/kg/day and decreased activity at all dose levels were noted in both sexes. Postdose observations were likely associated with peak ketonemia and were considered adverse at 15,000 mg/kg/day. These daily observations decreased over the study without any persistent effects, as determined during weekly pre-dose observations. Adverse histopathological changes included ulceration/erosion in non-glandular stomach at ≥ 12,000 mg/k/day and in glandular stomach at 15,000 mg/kg/day. These histopathological findings were not noted after 28-days of recovery. Due to unlikely human relevance of the rat non-glandular stomach effects for BH-BD and test substance-related mortality at 15,000 mg/kg/day, the no-observed-adverse-effect level (NOAEL) for subchronic toxicity of BH-BD was determined to be 12,000 mg/kg/day.

Equilibrative Nucleoside Transporters Mediate the Import of Nicotinamide Riboside and Nicotinic Acid Riboside into Human Cells.

Nicotinamide riboside (NR), a new form of vitamin B3, is an effective precursor of nicotinamide adenine dinucleotide (NAD+) in human and animal cells. The introduction of NR into the body effectively increases the level of intracellular NAD+ and thereby restores physiological functions that are weakened or lost in experimental models of aging and various pathologies. Despite the active use of NR in applied biomedicine, the mechanism of its transport into mammalian cells is currently not understood. In this study, we used overexpression of proteins in HEK293 cells, and metabolite detection by NMR, to show that extracellular NR can be imported into cells by members of the equilibrative nucleoside transporter (ENT) family ENT1, ENT2, and ENT4. After being imported into cells, NR is readily metabolized resulting in Nam generation. Moreover, the same ENT-dependent mechanism can be used to import the deamidated form of NR, nicotinic acid riboside (NAR). However, NAR uptake into HEK293 cells required the stimulation of its active utilization in the cytosol such as phosphorylation by NR kinase. On the other hand, we did not detect any NR uptake mediated by the concentrative nucleoside transporters (CNT) CNT1, CNT2, or CNT3, while overexpression of CNT3, but not CNT1 or CNT2, moderately stimulated NAR utilization by HEK293 cells.

In vitro stability and in vivo pharmacokinetics of the novel ketogenic ester, bis hexanoyl (R)-1,3-butanediol.

A novel ketone ester, bis hexanoyl (R)-1,3-butanediol (BH-BD), has been developed as a means to elevate blood ketones, for use as an energy substrate and a signaling metabolite. The metabolism of BH-BD and its effects on blood beta-hydroxybutyrate (BHB) levels was evaluated in various in vitro matrices and through analysis of plasma collected from Sprague Dawley rats and C57/BL6 mice in two oral gavage studies. A well-characterized ketone ester, (R)-3-hydroxybutyl (R)-3-hydroxybutyrate (HB-BHB), was used as an active control throughout. In vitro assay results demonstrated that BH-BD likely remains intact in the stomach and is hydrolyzed in the small intestine into hexanoate and (R)-1,3-butanediol. If absorbed intact, BH-BD is subject to hydrolysis by non-CYP enzymes in liver and esterases in plasma. If BH-BD reaches the lower intestine it is metabolized by gut flora. Plasma BHB delivery increased in a dose-dependent manner in rats and mice following oral administration of BH-BD. All doses of BH-BD were well tolerated. At doses over 3 g/kg, BHB delivery was similar between BH-BD and HB-BHB. The results of these studies support the hydrolysis of BH-BD into hexanoate and (R)-1,3-butanediol which are metabolized into BHB, delivering a well-tolerated, sustained and dose-dependent increase in plasma BHB in rodents.

Degradation of Extracellular NAD+ Intermediates in Cultures of Human HEK293 Cells.

Nicotinamide adenine dinucleotide (NAD) is an essential redox carrier, whereas its degradation is a key element of important signaling pathways. Human cells replenish their NAD contents through NAD biosynthesis from extracellular precursors. These precursors encompass bases nicotinamide (Nam) and nicotinic acid and their corresponding nucleosides nicotinamide riboside (NR) and nicotinic acid riboside (NAR), now collectively referred to as vitamin B3. In addition, extracellular NAD+ and nicotinamide mononucleotide (NMN), and potentially their deamidated counterparts, nicotinic acid adenine dinucleotide (NAAD) and nicotinic acid mononucleotide (NAMN), may serve as precursors of intracellular NAD. However, it is still debated whether nucleotides enter cells directly or whether they are converted to nucleosides and bases prior to uptake into cells. Here, we studied the metabolism of extracellular NAD+ and its derivatives in human HEK293 cells using normal and serum-free culture medium. Using medium containing 10% fetal bovine serum (FBS), mono- and dinucleotides were degraded to the corresponding nucleosides. In turn, the nucleosides were cleaved to their corresponding bases. Degradation was also observed in culture medium alone, in the absence of cells, indicating that FBS contains enzymatic activities which degrade NAD+ intermediates. Surprisingly, NR was also rather efficiently hydrolyzed to Nam in the absence of FBS. When cultivated in serum-free medium, HEK293 cells efficiently cleaved NAD+ and NAAD to NMN and NAMN. NMN exhibited rather high stability in cell culture, but was partially metabolized to NR. Using pharmacological inhibitors of plasma membrane transporters, we also showed that extracellular cleavage of NAD+ and NMN to NR is a prerequisite for using these nucleotides to maintain intracellular NAD contents. We also present evidence that, besides spontaneous hydrolysis, NR is intensively metabolized in cell culture by intracellular conversion to Nam. Our results demonstrate that both the cultured cells and the culture medium mediate a rather active conversion of NAD+ intermediates. Consequently, in studies of precursor supplementation and uptake, the culture conditions need to be carefully defined.

Medical and social legal aspects of the use of hydrophyte plants for food

The species diversity of hydrophytes suitable for human consumption and the possibility of their introduction into the practice of organic farming and sustainable polyculture are explored in the article. The economic and environmental potential of shallow freshwater areas and waterlogged areas are discussed from the perspective of sustainable agriculture. The possibility of using some hydrophyte plants for food and drugs is indicated. The necessity of using the practice of traditional nature management by the native population in relation to water and near-water food plants is mentioned. The relevant issues of providing the growing population of the Earth with food and technical plant raw materials from hydrophytes are discussed. It has been established that a necessary condition for the operation of the market for wild-growing medicinal hydrophytes in accordance with the concept of sustainable development is its efficient legal regulation at all levels.

Keeping the balance in NAD metabolism.

Research over the last few decades has extended our understanding of nicotinamide adenine dinucleotide (NAD) from a vital redox carrier to an important signalling molecule that is involved in the regulation of a multitude of fundamental cellular processes. This includes DNA repair, cell cycle regulation, gene expression and calcium signalling, in which NAD is a substrate for several families of regulatory proteins, such as sirtuins and ADP-ribosyltransferases. At the molecular level, NAD-dependent signalling events differ from hydride transfer by cleavage of the dinucleotide into an ADP-ribosyl moiety and nicotinamide. Therefore, non-redox functions of NAD require continuous biosynthesis of the dinucleotide. Maintenance of cellular NAD levels is mainly achieved by nicotinamide salvage, yet a variety of other precursors can be used to sustain cellular NAD levels via different biosynthetic routes. Biosynthesis and consumption of NAD are compartmentalised at the subcellular level, and currently little is known about the generation and role of some of these subcellular NAD pools. Impaired biosynthesis or increased NAD consumption is deleterious and associated with ageing and several pathologies. Insults to neurons lead to depletion of axonal NAD and rapid degeneration, partial rescue can be achieved pharmacologically by administration of specific NAD precursors. Restoring NAD levels by stimulating biosynthesis or through supplementation with precursors also produces beneficial therapeutic effects in several disease models. In this review, we will briefly discuss the most recent achievements and the challenges ahead in this diverse research field.

Genetic toxicity studies of glycolipids from Dacryopinax spathularia.

A series of in vitro studies were conducted to assess the genetic toxicity of jelly mushroom glycolipids from Dacryopinax spathularia (herein referred to as "AM-1"). In the bacterial reverse mutation assay (Ames test), there was no evidence of mutagenic activity in any Salmonella typhimurium strains tested or in Escherichia coli strain WP2uvrA, at dose levels up to 5000 µg/plate. In the micronucleus (MN) test using human lymphocytes, AM-1 did not show a statistically significant increase in the number of binucleated cells containing micronuclei when compared to concurrent control cultures at all time points and at any of the concentrations analyzed (up to 900 µg/ml of culture medium). No increase in mutation frequency or numbers of small and large colonies were noted for AM-1 (up to 800 µg/ml) compared to concurrent controls when tested in the mouse lymphoma thymidine kinase assay (MLA). Therefore, AM-1 was concluded to be negative in all three assays performed both in the absence and presence of Aroclor 1254- or phenobarbital/ß-naphthoflavone-induced rat liver (S9 mix) for metabolic activation. These results support the safety assessment of jelly mushroom glycolipids for potential use in food.

NAD Metabolome Analysis in Human Cells Using ¹H NMR Spectroscopy.

Nicotinamide adenine dinucleotide (NAD) and its phosphorylated form, NADP, are the major coenzymes of redox reactions in central metabolic pathways. Nicotinamide adenine dinucleotide is also used to generate second messengers, such as cyclic ADP-ribose, and serves as substrate for protein modifications including ADP-ribosylation and protein deacetylation by sirtuins. The regulation of these metabolic and signaling processes depends on NAD availability. Generally, human cells accomplish their NAD supply through biosynthesis using different forms of vitamin B3: Nicotinamide (Nam) and nicotinic acid as well as nicotinamide riboside (NR) and nicotinic acid riboside (NAR). These precursors are converted to the corresponding mononucleotides NMN and NAMN, which are adenylylated to the dinucleotides NAD and NAAD, respectively. Here, we have developed an NMR-based experimental approach to detect and quantify NAD(P) and its biosynthetic intermediates in human cell extracts. Using this method, we have determined NAD, NADP, NMN and Nam pools in HEK293 cells cultivated in standard culture medium containing Nam as the only NAD precursor. When cells were grown in the additional presence of both NAR and NR, intracellular pools of deamidated NAD intermediates (NAR, NAMN and NAAD) were also detectable. We have also tested this method to quantify NAD+ in human platelets and erythrocytes. Our results demonstrate that ¹H NMR spectroscopy provides a powerful method for the assessment of the cellular NAD metabolome.

Developmental and reproduction toxicity studies of glycolipids from Dacryopinax spathularia.

The developmental and reproduction toxicity potential of jelly mushroom glycolipids from Dacryopinax spathularia was studied in Crl:CD (SD) rats by daily oral gavage administration at doses of 150, 500 or 1000 mg/kg/day. Pregnant female rats in the developmental study received the test article from Gestation Days 6-19. F0 and F1 parental animals in the 2-generation reproduction toxicity study were dosed for a minimum of 70 days prior to mating and throughout mating, gestation, and lactation, until the day prior to euthanasia (following weaning of litters on postnatal day 21). The offspring of the F0 and F1 generations were potentially exposed to the test article in utero and via the milk while nursing. In the developmental study, there were no adverse effects on intrauterine growth and survival, or fetal morphology. In the 2-generation reproduction toxicity study, there were no adverse effects on observed parameters including macroscopic or microscopic findings, or organ weights for F0 or F1 animals, no effects on reproductive performance, and no test article-related effects on F1 and F2 postnatal survival, development, or growth. Therefore, the no-observed-adverse-effect level (NOAEL) for parental systemic toxicity, parental reproductive toxicity, and developmental/neonatal toxicity, was considered to be 1000 mg/kg/day, the highest dosage tested.

Pharmacokinetics, excretion balance, and tissue distribution of [14C]-labeled glycolipids and long chain fatty acids from Dacryopinax spathularia in rats.

The pharmacokinetics, excretion balance, and tissue distribution of [14C]-labeled glycolipids from Dacryopinax spathularia (herein referred to as "AM-1") and [14C]-LCFA equivalents following single or repeated administration to Sprague Dawley rats were evaluated to support the safety assessment of these naturally derived jelly mushroom glycolipids for use as a food ingredient. Rats received equimolar doses of either [14C]-AM-1 or [14C]-LCFA via oral or intravenous administration followed by collection of biological samples at specified intervals. Approximately 88%-101% of the administered dose was recovered in expired air, urine, feces, and carcass following single or repeated oral administration of [14C]-AM-1 at 100 mg/kg or equimolar doses of [14C]-LCFA at 46 mg/kg. Cmax and AUClast for [14C]-AM-1- and [14C]-LCFA-equivalents-derived radioactivity detected by quantitative whole body autoradiography was highest in the tissues of the GI tract, as expected following oral administration. The remaining tissues had low concentrations of test article equivalents relative to the administered dose and no target tissues for residence or accumulation were identified. AM-1 and LCFA are poorly absorbed by the oral route and are primarily eliminated in the feces without absorption. Oral bioavailability of both AM-1 and LCFA including their metabolites is low at approximately 11%.

Compartment-Specific Poly-ADP-Ribose Formation as a Biosensor for Subcellular NAD Pools.

Nicotinamide adenine dinucleotide (NAD) is vital to many cellular processes and is distributed between distinct subcellular pools in the compartmentalized eukaryotic cell. The detection and relative quantification of these individual pools is difficult because of the methods usually applied, which require cell disruption and fractionation.Here, we describe an immunochemical method to visualize and relatively quantify subcellular NAD+ pools, which relies on the NAD+-consuming activity of poly-ADP-ribose polymerase 1 (PARP1). We demonstrate that this system can be readily applied to detect changes in the mitochondrial, Golgi, endoplasmic reticulum, and peroxisomal NAD+ pools.

A 90-day oral toxicity study of glycolipids from Dacryopinax spathularia in Beagle dogs.

The subchronic toxicity of glycolipids from Dacryopinax spathularia (herein referred to as "AM-1") was studied in male and female Beagle dogs administered AM-1 by oral capsule at doses of 150, 500 or 1000 mg/kg/day for 90 days. AM-1 was well tolerated at all dosages and there were no test article-related effects on survival, clinical observations, neurological screening (functional observational battery) parameters, clinical pathology parameters, organ weights, macroscopic or microscopic evaluations. Test article-related changes were limited to minimal effects on food consumption and body weight changes in the 1000 mg/kg/day group females. Therefore, the no-observed-adverse-effect level (NOAEL) was considered to be 1000 mg/kg/day, the highest dosage level tested. These results add to the safety database for these naturally derived jelly mushroom glycolipids with potential for use as a food ingredient.