Cytotoxic effects of methanol, formaldehyde, and formate on dissociated rat thymocytes: A possibility of aspartame toxicity

Aspartame is a widely used artificial sweetener added to many soft beverages and its usage is increasing in health-conscious societies. Upon ingestion, this artificial sweetener produces methanol as a metabolite. In order to examine the possibility of aspartame toxicity, the effects of methanol and its metabolites (formaldehyde and formate) on dissociated rat thymocytes were studied by flow cytometry. While methanol and formate did not affect cell viability in the physiological pH range, formaldehyde at 1–3 mmol/L started to induce cell death. Further increase in formaldehyde concentration produced a dose-dependent decrease in cell viability. Formaldehyde at 1 mmol/L or more greatly reduced cellular content of glutathione, possibly increasing cell vulnerability to oxidative stress. Furthermore, formaldehyde at 3 mmol/L or more significantly increased intracellular concentration of Ca²⁺([Ca²⁺]_i) in a dose-dependent manner. Threshold concentrations of formaldehyde, a metabolite of methanol, that affected the [Ca²⁺]_iand cellular glutathione content were slightly higher than the blood concentrations of methanol previously reported in subjects administered abuse doses of aspartame. It is suggested that aspartame at abuse doses is harmless to humans. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effect of chronic exposure to aspartame on oxidative stress in brain discrete regions of albino rats

This study was aimed at investigating the chronic effect of the artificial sweetener aspartame on oxidative stress in brain regions of Wistar strain albino rats. Many controversial reports are available on the use of aspartame as it releases methanol as one of its metabolite during metabolism. The present study proposed to investigate whether chronic aspartame (75 mg/kg) administration could release methanol and induce oxidative stress in the rat brain. To mimic the human methanol metabolism, methotrexate (MTX)-treated rats were included to study the aspartame effects. Wistar strain male albino rats were administered with aspartame orally and studied along with controls and MTX-treated controls. The blood methanol level was estimated, the animal was sacrificed and the free radical changes were observed in brain discrete regions by assessing the scavenging enzymes, reduced glutathione, lipid peroxidation (LPO) and protein thiol levels. It was observed that there was a significant increase in LPO levels, superoxide dismutase (SOD) activity, GPx levels and CAT activity with a significant decrease in GSH and protein thiol. Moreover, the increases in some of these enzymes were region specific. Chronic exposure of aspartame resulted in detectable methanol in blood. Methanol per se and its metabolites may be responsible for the generation of oxidative stress in brain regions.     

Inhibition of CO2 production from aminopyrine or methanol by cyanamide or crotonaldehyde and the role of mitochondrial aldehyde dehydrogenase in formaldehyde oxidation

Previous results have shown that cyanamide or crotonaldehyde are effective inhibitors of the oxidation of formaldehyde by the low-Km mitochondrial aldehyde dehydrogenase, but do not affect the activity of the glutathione-dependent formaldehyde dehydrogenase. These compounds were used to evaluate the enzyme pathways responsible for the oxidation of formaldehyde generated during the metabolism of aminopyrine or methanol by isolated hepatocytes. Both cyanamide and crotonaldehyde inhibited the production of 14CO2 from 14C-labeled aminopyrine by 30-40%. These agents caused an accumulation of formaldehyde which was identical to the loss in CO2 production, indicating that the inhibition of CO2 production reflected an inhibition of formaldehyde oxidation. The oxidation of methanol was stimulated by the addition of glyoxylic acid, which increases the rate of H2O2 generation. Crotonaldehyde inhibited CO2 production from methanol, but caused a corresponding increase in formaldehyde accumulation. The partial sensitivity of CO2 production to inhibition by cyanamide or crotonaldehyde suggests that both the mitochondrial aldehyde dehydrogenase and formaldehyde dehydrogenase contribute towards the metabolism of formaldehyde which is generated from mixed-function oxidase activity or from methanol, just as both enzyme systems contribute towards the metabolism of exogenously added formaldehyde.     

The effects of aspartame on the HTR8/SVneo extravillous trophoblast cell line

Extravillous trophoblasts (EVTs) are a key cell type involved in placentation. Aspartame is an artificial sweetener with a widespread use. In rodents, aspartame ingestion during pregnancy was found to cause a reduction in placental and fetal weights, but its effect in placentation at a cellular level has not been studied. Aspartame is completely hydrolyzed in the gastrointestinal tract into L-phenylalanine, L-aspartic acid, and methanol. We aimed to study the effects of aspartame and its metabolites on placentation related characteristics of EVTs. For this, we exposed HTR-8/SVneo cells to aspartame (0.001, 0.01, 0.1, 0.5 and 1 mM), L-phenylalanine (0.14 and 0.5 mM), L-aspartic acid (0.82, 2.8 and 10 mM) or methanol (0.14 and 0.8 mM) for 24 h. Aspartame had an anti-proliferative effect, decreased the number of metabolically active cells and glucose cellular uptake and increased the number of cells arrested in S phase. L-aspartic acid significantly reduced glucose uptake and whole-cell protein content. L-phenylalanine had an anti-proliferative effect and increased the number of metabolically active cells. Interestingly, methanol exerted very marked effects on HTR8/SVneo cells: it showed an anti-proliferative effect, decreased glucose uptake, the migratory ability and the number of cells in the G2/M phase and increased oxidative stress levels, in concentrations corresponding to the blood levels after the 99th percentile of projected daily ingestion of aspartame. Overall, our results demonstrate that aspartame and its metabolites can affect several characteristics of EVTs and support the conclusion that the effect of aspartame in the placenta should be further evaluated.     

Pixantrone can be activated by formaldehyde to generate a potent DNA adduct forming agent

Mitoxantrone is an anti-cancer agent used in the treatment of breast and prostate cancers. It is classified as a topoisomerase II poison, however can also be activated by formaldehyde to generate drug-DNA adducts. Despite identification of this novel form of mitoxantrone-DNA interaction, excessively high, biologically irrelevant drug concentrations are necessary to generate adducts. A search for mitoxantrone analogues that could potentially undergo this reaction with DNA more efficiently identified Pixantrone as an ideal candidate. An in vitro crosslinking assay demonstrated that Pixantrone is efficiently activated by formaldehyde to generate covalent drug-DNA adducts capable of stabilizing double-stranded DNA in denaturing conditions. Pixantrone-DNA adduct formation is both concentration and time dependent and the reaction exhibits an absolute requirement for formaldehyde. In a direct comparison with mitoxantrone-DNA adduct formation, Pixantrone exhibited a 10- to 100-fold greater propensity to generate adducts at equimolar formaldehyde and drug concentrations. Pixantrone-DNA adducts are thermally and temporally labile, yet they exhibit a greater thermal midpoint temperature and an extended half-life at 37 degrees C when compared to mitoxantrone-DNA adducts. Unlike mitoxantrone, this enhanced stability, coupled with a greater propensity to form covalent drug-DNA adducts, may endow formaldehyde-activated Pixantrone with the attributes required for Pixantrone-DNA adducts to be biologically active.     

Barminomycin functions as a potent pre-activated analogue of Adriamycin.

The anthracycline Adriamycin is known to form adducts with DNA, but requires prior activation by formaldehyde. In contrast, the anthracycline barminomycin is also able to form adducts with DNA, but does not require activation by formaldehyde. Barminomycin, therefore, appears to function as a pre-activated form of Adriamycin. The DNA adducts formed by both anthracyclines are bound covalently to only one strand of DNA, but both also stabilise duplex DNA sufficiently that they can be detected as virtual interstrand crosslinks in heat denaturation electrophoretic crosslinking assays. The barminomycin-DNA adducts form extremely rapidly with DNA, and at exceedingly low concentrations (approximately 50-fold lower than with Adriamycin in the presence of excess formaldehyde), both characteristics consistent with barminomycin being in a pre-activated state, hence, undergoing a bimolecular reaction with DNA compared with the trimolecular reaction (drug, formaldehyde and DNA) required with Adriamycin. Surprisingly, barminomycin-DNA adducts are substantially more stable (essentially irreversible) than Adriamycin-DNA adducts (half life of approximately 25 h at 37 degrees C). Due to this understanding of the reactivity of barminomycin and its exceptional cytotoxicity (1000-fold more cytotoxic than Adriamycin), detailed structural studies of barminomycin-DNA adducts are now warranted, both in vitro and in tumour cells.     

Formaldehyde-activated WEHI-150 induces DNA interstrand crosslinks with unique structural features

Mitoxantrone is an anticancer anthracenedione that can be activated by formaldehyde to generate covalent drug-DNA adducts. Despite their covalent nature, these DNA lesions are relatively labile. It was recently established that analogues of mitoxantrone featuring extended side-chains terminating in primary amino groups typically yielded high levels of stable DNA adducts following their activation by formaldehyde. In this study we describe the DNA sequence-specific binding properties of the mitoxantrone analogue WEHI-150 which is the first anthracenedione to form apparent DNA crosslinks mediated by formaldehyde. The utility of this compound lies in the versatility of the covalent binding modes displayed. Unlike other anthracenediones described to date, WEHI-150 can mediate covalent adducts that are independent of interactions with the N-2 of guanine and is capable of adduct formation at novel DNA sequences. Moreover, these covalent adducts incorporate more than one formaldehyde-mediated bond with DNA, thus facilitating the formation of highly lethal DNA crosslinks. The versatility of binding observed is anticipated to allow the next generation of anthracenediones to interact with a broader spectrum of nucleic acid species than previously demonstrated by the parent compounds, thus allowing for more diverse biological activities.     

Neuropsychological and biochemical investigations in heterozygotes for phenylketonuria during ingestion of high dose aspartame (a sweetener containing phenylalanine)

Aspartame, a high intensity sweetener, is used extensively worldwide in over 5,000 products. Upon ingestion, aspartame is completely metabolized to two amino acids and methanol (approximately 50% phenylalanine, 40% aspartic acid, and 10% methanol). The effects of aspartame on cognitive function, electroencephalograms (EEGs) and biochemical parameters were evaluated in 48 adult (21 men, 27 women) heterozygotes for phenylketonuria (PKUH). PKUH subjects whose carrier status had been proven by DNA analysis ingested aspartame (either 15 or 45 mg/kg/day) and placebo for 12 weeks on each treatment using a randomized, doubleblind, placebo-controlled, crossover study. A computerized battery of neuropsychological tests was administered at baseline weeks -2 and -1, and during treatment at weeks 6, 12, 18, and 24. Samples for plasma amino acids and urinary organic acids were also collected during these visits. EEGs were evaluated by conventional and spectral analysis at baseline week -1 and treatment weeks 12 and 24. The results of the neuropsychological tests demonstrated that aspartame had no effect on cognitive function. Plasma phenylalanine significantly increased, within the normal range for PKUH, at 1 and 3 h following the morning dose of aspartame in the group receiving the 45 mg/kg per day dose only. There were no significant differences in the conventional or spectral EEG analyses, urinary organic acid concentrations, and adverse experiences when aspartame was compared with placebo. This study reaffirms the safety of aspartame in PKUH and refutes the speculation that aspartame affects cognitive performance, EEGs, and urinary organic acids.     

The power and potential of doxorubicin-DNA adducts

Doxorubicin (trade name Adriamycin) is a widely used anticancer agent which exhibits good activity against a wide range of tumors. Although the major mode of action appears to be normally as a topoisomerase II poison, it also exhibits a number of other cellular responses, one of which is the ability to form adducts with DNA. For adduct formation doxorubicin must react with cellular formaldehyde to form an activated Schiff base which is then able to form an aminal (N-C-N) linkage to the exocyclic amino group of guanine residues. The mono-adducts form primarily at G of 5'-GCN-3' sequences where the chromophore of the drug is intercalated between the C and N base pair. The structure of the adducts has have been well defined by 2D NMR, mass spectrometry and X-ray crystallography. The formation of these anthracycline adducts in cells grown in culture has been unequivocally demonstrated. The source of formaldehyde in cells can be endogenous, provided by coadministration of prodrugs that release formaldehyde or by prior complexation of anthracyclines with formaldehyde. Since the adducts appear to be more cytotoxic than doxorubicin alone, and also less susceptible to drug-efflux forms of resistance, they offer new approaches to improving the anticancer activity of the anthracyclines.     

Link between the Membrane-Bound Pyridine Nucleotide Transhydrogenase and Glutathione-Dependent Processes in Rhodobacter sphaeroides

The presence of a glutathione-dependent pathway for formaldehyde oxidation in the facultative phototroph Rhodobacter sphaeroides has allowed the identification of gene products that contribute to formaldehyde metabolism. Mutants lacking the glutathione-dependent formaldehyde dehydrogenase (GSH-FDH) are sensitive to metabolic sources of formaldehyde, like methanol. This growth phenotype is correlated with a defect in formaldehyde oxidation. Additional methanol-sensitive mutants were isolated that contained Tn5 insertions in pntA, which encodes the alpha subunit of the membrane-bound pyridine nucleotide transhydrogenase. Mutants lacking transhydrogenase activity have phenotypic and physiological characteristics that are different from those that lack GSH-FDH activity. For example, cells lacking transhydrogenase activity can utilize methanol as a sole carbon source in the absence of oxygen and do not display a formaldehyde oxidation defect, as determined by whole-cell (13)C-nuclear magnetic resonance. Since transhydrogenase can be a major source of NADPH, loss of this enzyme could result in a requirement for another source for this compound. Evidence supporting this hypothesis includes increased specific activities of other NADPH-producing enzymes and the finding that glucose utilization by the Entner-Doudoroff pathway restores aerobic methanol resistance to cells lacking transhydrogenase activity. Mutants lacking transhydrogenase activity also have higher levels of glutathione disulfide under aerobic conditions, so it is consistent that this strain has increased sensitivity to oxidative stress agents like diamide, which are known to alter the oxidation reduction state of the glutathione pool. A model will be presented to explain the role of transhydrogenase under aerobic conditions when cells need glutathione both for GSH-FDH activity and to repair oxidatively damaged proteins.     

Sodium ions and an energized membrane required by Methanosarcina barkeri for the oxidation of methanol to the level of formaldehyde.

Methanogenesis from methanol by cell suspensions of Methanosarcina barkeri was inhibited by the uncoupler tetrachlorosalicylanilide. This inhibition was reversed by the addition of formaldehyde. 14C labeling experiments revealed that methanol served exclusively as the electron acceptor, whereas formaldehyde was mainly oxidized to CO2 under these conditions. These data support the hypothesis (M. Blaut and G. Gottschalk, Eur. J. Biochem. 141: 217-222, 1984) that the first step in methanol oxidation depends on the proton motive force or a product thereof. Cell extracts of M. barkeri converted methanol and formaldehyde to methane under an H2 atmosphere. Under an N2 atmosphere, however, formaldehyde was disproportionated to CH4 and CO2, whereas methanol was metabolized to a very small extent only, irrespective of the presence of ATP. It was concluded that cell extracts of M. barkeri are not able to oxidize methanol. In further experiments, the sodium dependence of methanogenesis and ATP formation by whole cells was investigated. Methane formation from methanol alone and the corresponding increase in the intracellular ATP content were strictly dependent on Na+. If, in contrast, methanol was utilized together with H2, methane and ATP were synthesized in the absence of Na+. The same is true for the disproportionation of formaldehyde to methane and carbon dioxide. From these experiments, it is concluded that in M. barkeri, Na+ is involved not in the process of ATP synthesis but in the first step of methanol oxidation.     

Methanol metabolism in the Asian corn borer, Ostrinia furnacalis (Guenée) (Lepidoptera: Pyralidae)

Plants produce and release large quantities of methanol, especially when attacked by herbivores. It seems that the herbivores may suffer from methanol intoxication. Here we reported the tolerance to and the metabolism of methanol by Ostrinia furnacalis third-instar larvae. When larvae were exposed to dietary methanol, formaldehyde and formic acid for 72 h, the estimated LC₅₀ value was 28, 40 and 29 mg/g diet, respectively. Toxicity of methanol was enhanced by 4-methylpyrazole, 3-amino-1,2,4-triazole and piperonyl butoxide, and toxicity of formaldehyde was increased by 3-amino-1,2,4-triazole and piperonyl butoxide. However, triphenyl phosphate had little synergistic effects on both methanol and formaldehyde. These data indicate that alcohol dehydrogenase, and probably catalase and cytochrome P450 monooxygenase oxidize methanol to formaldehyde, catalase and cytochrome P450 monooxygenase catalyze formaldehyde to formic acid, water and carbon dioxide, and carboxylesterase may have a minor effect. Several fatty acid methyl esters (FAMEs) were identified from extracts of the frass of larvae which had been exposed to a methanol-contained diet, in contrast to those on a methanol-free artificial diet. In vitro tests revealed that a crude enzyme solution from the larvae could synthesize FAMEs from corresponding fatty acids and methanol. In addition, dietary methanol induced higher esterase activities in the first-, second- and third-instar larvae. These findings demonstrate that both oxidative metabolism and non-oxidative metabolism are partially responsible for methanol elimination in O. furnacalis larvae.     

How overproduction of foreign proteins affects physiology of the recombinant strains ofHansenula polymorpha

Changes in the activity of key enzymes of the methanol utilization pathway of the recombinant strains of methylotrophic yeastHansenula polymorpha R22-2B and LAC-56 were studied at different rates of chemostat growth on methanol containing mineral media. It was shown that the strain R22-2B, initially having a 10-fold increased activity of dihydroxyacetone kinase (DHAK, a key enzyme of formaldehyde assimilation) acquired increased activity of formaldehyde dehydrogenase (FADH, a key enzyme of formaldehyde dissimilation) which resulted in the enhanced oxidation of formaldehyde to CO₂. Strain LAC-56, overproducingEscherichia coli β-galactosidase, acquired the decreased intracellular concentration of ATP which resulted in the decrease of the efficiency of formaldehyde assimilation catalyzed by DHAK and resulted in accumulation of toxic formaldehyde. As a consequence some biochemical responses occurred in cells that were directed to a diminishing of the toxic effect of accumulated formaldehyde, namely, the decreasing of methanol oxidase activity (to reduce the rate of formaldehyde synthesis), and the increasing of FADH activity (to increase the rate of formaldehyde oxidation).     

Conversion of methanol to formic acid through the coupling of the enzyme reactions of alcohol oxidase,catalase and formaldehyde dismutase

Summary A novel process for the production of formic acid from methanol has been developed that involves the coupled reactions of the three enzymes, alcohol oxidase, catalase and formaldehyde dismutase. In this process, methanol is oxidized to formaldehyde by alcohol oxidase and catalase, followed by the formaldehyde dismutase reaction that leads to the formation of methanol and formic acid. Ultimately, the substrate methanol (100 to 200 mM) is completely converted to formic acid, by the recycling of the consecutive enzyme reactions.     

Formation of DNA adducts by formaldehyde-activated mitoxantrone.

Recent studies with the anthracycline Adriamycin have demonstrated its activation by formaldehyde and subsequent binding to DNA in vitro. Since formaldehyde levels are known to be higher in cells of myeloid origin and the structurally related drug mitoxantrone is most effective against cancers of myeloid origin, this indicates a possible role of formaldehyde in the activation of mitoxantrone. In vitro studies revealed that the activation of mitoxantrone by formaldehyde leads to the formation of drug-DNA adducts. These adducts stabilised DNA such that they functioned as virtual interstrand crosslinks. The interstrand crosslinks were formed in the presence of mitoxantrone and formaldehyde in a time- and concentration-dependent manner. In the absence of formaldehyde no crosslinks were formed, indicating a key role in drug activation and DNA binding. The adducts (virtual crosslinks) were relatively unstable with 50% crosslinks remaining after 10 min at 60 degrees C in 45% formamide. Like Adriamycin, the mitoxantrone-formaldehyde-DNA crosslinks are heat labile and do not display the stability associated with covalent interstrand crosslinks.     

Formaldehyde incorporation by a new methylotroph (L3)

A number of bacterial strains have been isolated and investigated in our search for a promising organism in the production of single-cell protein from methanol. Strain L3 among these isolates was identified as an obligate methylotroph which grew only on methanol and formaldehyde as the sole sources of carbon and energy. The organism also grew well in batch and chemostat mixed-substrate cultures containing methanol, formaldehyde, and formate. Although formate was not utilized as a sole carbon and energy source, it was readily taken up and oxidized by either formaldehyde- or methanol-grown cells. The organism incorporated carbon by means of the ribulose monophosphate pathway when growing on either methanol, formaldehyde, or various mixtures of C1 compounds. Its C1-oxidation enzymes included phenazine methosulfate-linked methanol and formaldehyde dehydrogenase and a nicotinamide adenine dinucleotide-linked formate dehydrogenase. Identical inhibition by formaldehyde of the first two dehydrogenases suggested that they are actually the same enzyme. The organism had a rapid growth rate, a high cell yield in the chemostat, a high protein content, and a favorable amino acid distribution for use as a source of single-cell protein. Of special interest was the ability of the organism to utilize formaldehyde via the ribulose monophosphate cycle.     

Regulation phenomena in methanol consuming yeasts: An experiment with model discrimination

Assimilation as well as dissimilation of methanol in yeasts takes place through its oxidative intermediate formaldehyde which is several times more toxic to the growth of microorganisms than methanol itself. Still, the role of formaldehyde, produced during methanol assimilation, upon growth of yeasts is not clear. In the present paper, an attempt has been made to throw some light upon this aspect. Starting with a basic frame work for methanol uptake by yeasts, several models were developed assuming different modes of regulation of key enzymes by methanol and/or formaldehyde. The main feature of the basic framework consists in consideration of two routes for oxidation of formaldehyde to CO₂, one associated and the other not associated with production of energy. Further, the rate of energy production form the energy-associated oxidation of formaldehyde is assumed to be controlled by the rate of energy consumption by anabolic reactions. The models were discriminated by subjecting these to biological constraints. As a result, the successful model suggests that in spite of higher inherent toxicity of formaldehyde, methanol exerts the controlling influence upon growth under normal conditions.