Nitrosation of aspartic acid, aspartame, and glycine ethylester. Alkylation of 4-(p-nitrobenzyl)pyridine (NBP) in vitro and binding to DNA in the rat

Meier, Irene B.; Shephard, Sarah E.; Lutz, Werner

doi:10.1016/0165-1110(90)90011-y

Cited by 15 publications

(19 citation statements)

References 10 publications

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“…An alkylation reaction of the jujube seed extracts was performed to understand the interactions of the extracts with DNA resulting in DNA damage, by testing their alkylating activity. The nitrobenzylpyridine (NBP) assay was used as a model for nucleophilic DNA bases [ 43 – 45 ]. This reaction depends on high temperature and low acidic media to catalyze NBP, which is converted into a blue/violet chromophore product.…”

Section: Resultsmentioning

confidence: 99%

Apoptosis-inducing effects of jujube (Zǎo) seed extracts on human Jurkat leukemia T cells

et al. 2016

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BackgroundJujube (Zǎo) seeds exhibited anticancer effects and used in Chinese medicine for many years. This study aims to investigate the apoptosis-inducing effects of seed extracts from eight different cultivated species (‘Apple’, ‘Bombay’, ‘Jumbo’, ‘Kaew’, ‘Nomsod’, ‘Rianthong’, ‘Samros’, and ‘Taiwan’) on human Jurkat leukemia T cells.MethodsWe evaluated the effects of seed extracts from eight jujube cultivated species on human Jurkat leukemia T cells. The crude seed extracts were prepared sequentially by using water, 95 % ethanol, dichloromethane, ethyl acetate, chloroform or hexane. The antiproliferative effects of the jujube seed extracts relative to that of melphalan were evaluated by neutral red assays. Apoptotic cell death induced by the ethanolic extracts at 1 × IC50 and 2 × IC50 concentrations was demonstrated by DAPI staining, gel electrophoresis, flow cytometry with Annexin V/propidium iodide staining, and caspase-3, -8, and -9 enzyme activities.ResultsEthanolic extracts of ‘Taiwan’, ‘Jumbo’, ‘Nomsod’, ‘Rianthong’, ‘Samros’, and ‘Bombay’, significantly inhibited the proliferation of Jurkat cells compared with untreated cells (all P < 0.001), while the extracts of ‘Kaew’ and ‘Apple’ were inactive. The six active extracts preferentially induced apoptotic cell death in a concentration-dependent manner with DNA fragmentation (2 × IC50). Increased caspase-3 activity was detected after treatment with the six extracts. The ‘Taiwan’, ‘Nomsod’, ‘Jumbo’, and ‘Rianthong’ extracts (2 × IC50) induced both the extrinsic and intrinsic apoptosis pathways by increasing caspase-8 and caspase-9 activity, respectively. Alkaloids (Dragendorff’s method) and reducing sugars (Fehling’s test) were mainly identified in the apoptosis-inducing extracts.ConclusionsThe tested of six active extracts (‘Taiwan’, ‘Jumbo’, ‘Nomsod’, ‘Rianthong’, ‘Samros’ and ‘Bombay’) contained alkaloids or reducing sugars, and induced caspase-dependent apoptosis in human Jurkat leukemia T cells.

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Section: Resultsmentioning

confidence: 99%

Apoptosis-inducing effects of jujube (Zǎo) seed extracts on human Jurkat leukemia T cells

et al. 2016

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“…The nitrosatable agent may be a metabolite which is present endogenously or has been released from cells into the growth medium. Possible candidates include the catabolites MA and MU, small peptides, and amino acids and their degradation products, all of which can be nitrosated to give directly acting mutagenic agents (9,29,49).…”

Section: Discussionmentioning

confidence: 99%

Generation of an endogenous DNA-methylating agent by nitrosation in Escherichia coli

Taverna¹,

Sedgwick²

1996

J Bacteriol

144

113

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Escherichia coli ada ogt mutants, which are totally deficient in O 6 -methylguanine-DNA methyltransferases, have an increased spontaneous mutation rate. This phenotype is particularly evident in starving cells and suggests the generation of an endogenous DNA alkylating agent under this growth condition. We have found that in wild-type cells, the level of the inducible Ada protein is 20-fold higher in stationary-phase and starving cells than in rapidly growing cells, thus enhancing the defense of these cells against DNA damage. The increased level of Ada in stationary cells is dependent on RpoS, a stationary-phase-specific sigma subunit of RNA polymerase. We have also identified a potential source of the mutagenic agent. Nitrosation of amides and related compounds can generate directly acting methylating agents and can be catalyzed by bacterial enzymes. E. coli moa mutants, which are defective in the synthesis of a molybdopterin cofactor required by several reductases, are deficient in nitrosation activity. It is reported here that a moa mutant shows reduced generation of a mutagenic methylating agent from methylamine (or methylurea) and nitrite added to agar plates. Moreover, a moa mutation eliminates much of the spontaneous mutagenesis in ada ogt mutants. These observations indicate that the major endogenous mutagen is not S-adenosylmethionine but arises by bacterially catalyzed nitrosation.Only a few types of cancer have been attributed to environmental agents. Other carcinogens of primary concern may be transient but highly reactive metabolites that arise during normal metabolism (24, 36). Indeed, several metabolites of amino acids, sugars, oxygen, and lipids are known to be mutagenic. It is, therefore, important to identify metabolites that damage DNA in vivo and result in spontaneous mutagenesis in the absence of adequate DNA repair. For example, reactive oxygen species generated during aerobic metabolism damage DNA bases and sugar residues. Specific oxygen radical detoxification and DNA repair mechanisms prevent or remove this damage (13,14). The increase in the rate of spontaneous mutagenesis in bacteria and yeast mutants that are specifically deficient in the repair of aberrantly methylated DNA bases indicates that methylation of DNA also occurs endogenously (16,34,52,53). Rodent cells may be similarly affected (2).The major mutagenic adduct induced in DNA by methylating agents is O 6 -methylguanine (O 6 -meG). This modified base mispairs with thymine during DNA replication, resulting in GC-to-AT transition mutations. In wild-type cells, O 6 -meG is repaired by ubiquitous O 6 -meG-DNA methyltransferases that directly transfer the methyl group from the modified base to one of their own cysteine residues. As a result of this process, the methyltransferases are inactivated (25). Escherichia coli has two O 6 -meG-DNA methyltransferases, a constitutive Ogt protein and an inducible Ada protein. Each exponentially growing cell contains only two to four molecules of Ada protein and 10-fold more Ogt protein (32,3...

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“…The Panel considered these findings, obtained in an acellular system in presence of excess aspartame, of minimal relevance for the evaluation of the genotoxic potential of aspartame. Meier et al (1990) investigated the kinetics of formation, stability and reactivity of nitrosation products of aspartic acid, aspartame, and glycine ethyl ester. Nitrosation products were obtained in vitro, with incubation of 40 mM substrate and nitrite at pH 2.5 for varying times.…”

Section: In Vivo Studiesmentioning

confidence: 99%

“…Concerning the studies of Meier et al (1990) and Shephard et al (1993), the Panel noted the harsh conditions utilised for the in vitro nitrosation of substrates and considered the results of doubtful relevance for the assessment of the genotoxic risk posed by the dietary intake of aspartame or other natural amino acids and dipeptides.…”

Section: In Vivo Studiesmentioning

confidence: 99%

Scientific Opinion on the re‐evaluation of aspartame (E 951) as a food additive

2013

EFS2

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The EFSA ANS Panel provides a scientific opinion on the safety of aspartame (E 951). Aspartame is a sweetener authorised as a food additive in the EU. In previous evaluations by JECFA and the SCF, an ADI of 40 mg/kg bw/day was established based on chronic toxicity in animals. Original reports, previous evaluations, additional literature and data made available following a public call were evaluated. Aspartame is rapidly and completely hydrolysed in the gastrointestinal tract to phenylalanine, aspartic acid and methanol. Chronic and developmental toxicities were relevant endpoints in the animal database. From chronic toxicity studies in animals, a NOAEL of 4000 mg/kg bw/day was identified. The possibility of developmental toxicity occurring at lower doses than 4000 mg/kg in animals could not be excluded. Based on MoA and weight‐of‐evidence analysis, the Panel concluded that developmental toxicity in animals was attributable to phenylalanine. Phenylalanine at high plasma levels is known to cause developmental toxicity in humans. The Panel concluded that human data on developmental toxicity were more appropriate for the risk assessment. Concentration‐response modelling was used to determine the effects of aspartame administration on plasma phenylalanine using human data after phenylalanine administration to normal, PKU heterozygote or PKU homozygote individuals. In normal and PKU heterozygotes, aspartame intakes up to the ADI of 40 mg/kg bw/day, in addition to dietary phenylalanine, would not lead to peak plasma phenylalanine concentrations above the current clinical guideline for the prevention of adverse effects in fetuses. The Panel concluded that aspartame was not of safety concern at the current aspartame exposure estimates or at the ADI of 40 mg/kg bw/day. Therefore, there was no reason to revise the ADI of aspartame. Current exposures to aspartame ‐ and its degradation product DKP ‐ were below their respective ADIs. The ADI is not applicable to PKU patients.

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Nitrosation of aspartic acid, aspartame, and glycine ethylester. Alkylation of 4-(p-nitrobenzyl)pyridine (NBP) in vitro and binding to DNA in the rat

Cited by 15 publications

References 10 publications

Apoptosis-inducing effects of jujube (Zǎo) seed extracts on human Jurkat leukemia T cells

Apoptosis-inducing effects of jujube (Zǎo) seed extracts on human Jurkat leukemia T cells

Generation of an endogenous DNA-methylating agent by nitrosation in Escherichia coli

Scientific Opinion on the re‐evaluation of aspartame (E 951) as a food additive

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