A mitochondrial chemiluminescence evoked by a novel mixed copper(II)-cyanide complex/acetaldehyde cyanohydrin chelate: a kinetic analysis suggesting a role for membrane-bound vicinal sulfhydryls

Cyanide added to mitochondria in the presence of copper and acetaldehyde evokes a chemiluminescence which follows series pseudo-first-order kinetics: (formula; see text) An evaluation of the effects of protein (mitochondria), copper, cyanide, acetaldehyde, and oxygen on the kinetic parameters shows that k1 is influenced by protein, cyanide (at low concentrations), and oxygen while k2 is influenced by cyanide, acetaldehyde (at low, less than 11.9 mM, and high, greater than 35.6 mM, concentrations), and oxygen. The integral light increases linearly with the square root of total copper(II) and with the square of the total acetaldehyde. The sustained emissions appear to reflect an initial oxidative event mediated by a novel mixed copper(II)-cyanide complex/acetaldehyde cyanohydrin chelate. Cu(I) formed by the reduction of Cu(II), probably by mitochondrial vicinal sulfhydryls, reacts with dioxygen to form an O2-copper complex which reacts with acetaldehyde to form the acetyl-1-hydroxyhydroperoxyl radical. This radical disproportionates by the Russell mechanism to generate electronically excited singlet and triplet carbonyl functions and singlet oxygen species whose emissive relaxations to the ground state display as the observed chemiluminescence. The kinetic evidence indicates that there are two Cu(I)-oxygen cyanide complexes transferring O2- to acetaldehyde. This evidence addresses the mechanisms of autoxidation of low-molecular-weight Cu(I) complexes with dioxygen. A suggested role for the involvement of vicinal sulfhydryl groups in the reactions is shown, kinetically, by the influence of copper and acetaldehyde on the integral light.     

Inhibition and stimulation of yeast growth by acetaldehyde

Summary Acetaldehyde at above about 0.3 g/l inhibited yeast growth, suggesting that it may contribute to product inhibition in alcohol fermentations when present at high concentrations intracellularly. The toxic effects of acetaldehyde and ethanol were not mutually reinforcing, acetaldehyde appearing to alleviate slightly the effects of ethanol. In support of this, low concentrations of acetaldehyde greatly reduced the lag phase in ethanol-containing medium and increased the specific growth rate.     

Ethanol-induced injuries to carrot cells : the role of acetaldehyde

Carrot (Daucus carota L.) cell cultures show high sensitivity to ethanol since both unorganized cell growth and somatic embryogenesis are strongly inhibited by ethanol at relatively low concentrations (10-20 millimolar). The role of acetaldehyde on ethanol-induced injuries to suspension cultured carrot cells was evaluated. When ethanol oxidation to acetaldehyde is prevented by adding an alcohol-dehydrogenase (EC 1.1.1.1) inhibitor (4-methylpyrazole) to the culture medium, no ethanol toxicity was observed, even if ethanol was present at relatively high concentrations (40-80 millimolar). Data are also presented on the effects of exogenously added acetaldehyde on both carrot cell growth and somatic embryogenesis. We conclude that the observed toxic effects of ethanol cannot be ascribed to ethanol per se but to acetaldehyde.     

Mitochondrial chemiluminescence elicited by acetaldehyde

Acetaldehyde-dependent chemiluminescence has been found to be a sensitive technique for the study of superoxide and hydrogen peroxide formation in beef heart mitochondria. The system responds to ATP and antimycin A with increased emission intensities and to ADP and rotenone with decreased intensities, indicating that the chemiluminescence reflects the energy status of the mitochondrion. These effects are based on the ability of acetaldehyde to react with superoxide and hydrogen peroxide to form metastable intermediates which decay spontaneously with the emission of light. Additionally, these intermediates can react with cyanide to give alternative products which can also decay with the emission of light, the cyanide-evokable chemiluminescence. The interaction of acetaldehyde with mitochondria is complex because acetaldehyde can serve as a hydrogen source for NADH and as an inhibitor (at high concentration) of electron transport, and appears to be a reducing agent for a heat-stable site that autoxidatively generates HOOH from O₂ ^–·. Inasmuch as acetaldehyde is a metabolite of ethanol, this broad spectrum of reactivity may play a role in the hepatic and cardiac toxicity that is associated with alcoholism. The heat-stable site that generates HOOH from O₂ ^–· has been studied further and appears to contain vicinal dithiol which is primarily responsible for the cyanide-evokable chemiluminescence.The work reported in this paper was carried out by Erin E. Boh in partial fulfillment of the requirements for the Doctor of Philosophy degree.     

Production of Acetaldehyde by Zymomonas mobilis

Mutants of Zymomonas mobilis were selected for decreased alcohol dehydrogenase activity by using consecutively higher concentrations of allyl alcohol. A mutant selected by using 100 mM allyl alcohol produced acetaldehyde at a level of 4.08 g/liter when the organism was grown in aerated batch cultures on a medium containing 4.0% (wt/wt) glucose. On the basis of the amount of glucose utilized, this level of acetaldehyde production represents nearly 40% of the maximum theoretical yield. Acetaldehyde produced during growth was continuously air stripped from the reactor. Acetaldehyde present in the exhaust stream was then trapped as the acetaldehyde-bisulfite addition product in an aqueous solution of sodium bisulfite and released by treatment with base. Acetaldehyde was found to inhibit growth of Z. mobilis at concentrations as low as 0.05% (wt/wt) acetaldehyde. An acetaldehyde-tolerant mutant of Z. mobilis was isolated after both mutagenesis with nitrosoguanidine and selection in the presence of vapor-phase acetaldehyde. The production of acetaldehyde has potential advantages over that of ethanol: lower energy requirements for product separation, efficient separation of product from dilute feed streams, continuous separation of product from the reactor, and a higher marketplace value.     

Impact of acetaldehyde- and pyruvic acid-bound sulphur dioxide on wine lactic acid bacteria

Aims: To investigate the impact of acetaldehyde‐ and pyruvic acid‐bound sulphur dioxide on wine lactic acid bacteria (LAB). Methods and Results: Growth studies were performed where Oenococcus oeni, Pediococcus parvulus, Ped. damnosus and Lactobacillus hilgardii were inoculated into media containing various concentrations of acetaldehyde or pyruvic acid and an equimolar concentration of SO₂ at pH 3·50 and 3·70. Low concentrations of acetaldehyde‐ and pyruvic acid‐bound SO₂ were inhibitory to the growth of all bacteria although acetaldehyde‐bound SO₂ was generally more inhibitory than pyruvic acid‐bound SO₂. Inhibition was greater at pH 3·50 than 3·70, and Lact. hilgardii was the most sensitive to acetaldehyde‐bound SO₂, while O. oeni was the most sensitive to pyruvic acid‐bound SO₂. Degradation of SO₂‐bound acetaldehyde was observed for all LAB, and aside from O. oeni, there was also complete degradation of SO₂‐bound pyruvic acid at both pH values. O. oeni only degraded pyruvic acid at pH 3·70. Degradation of SO₂‐bound acetaldehyde or pyruvic acid did not correlate with bacterial growth as inhibition was always observed in media containing bound SO₂. Conclusions: Acetaldehyde‐ and pyruvic acid‐bound SO₂ were inhibitory to wine LAB growth at concentrations as low as 5 mg l^?1. Despite this inhibition, all wine LAB degraded SO₂‐bound acetaldehyde and pyruvic acid suggesting that bound SO₂ may have a bacteriostatic rather than bacteriocidal action. Significance and Impact of the Study: Sulphur dioxide bound to acetaldehyde or pyruvic acid is inhibitory to growth of wine LAB and must be considered when conducting the malolactic fermentation or controlling the growth of spoilage bacteria such as Pediococcus and Lactobacillus.     

Chemiluminescence of daphnia cultivating medium and optimization of conditions for its determination

The article deals with the optimization of conditions for the chemiluminescence determination. The Daphnia habitat was shown to have no spontaneous chemiluminescence. This was revealed using hydrogen peroxide and luminol, the optimal concentrations of which were 23 and 1.6 x 10(-2) mmol/L. p-Iodphenol at low concentrations (4 x 10(-5)-2 x 10(-3) mmol/L) did not render its effect chemiluminescence signal while at high concentrations (4 x 10(-2) mmol/L) an inhibition of chemiluminescence was observed. To obtain the needed intensity of chemiluminescence no more than 5 daphnia persons is required to incubate in volume of 10 mL of sample for analyzing. The intensity of chemiluminescence of daphnia cultivating medium and the sensitivity of this organism to potassium chromate increased at the temperature increasing from 24 to 32 degrees C. Daphnia cultivating medium can be preserved in refrigerator for several hours without lost of chemiluminescence signal.     

Teratogenicity of acetaldehyde invitro: Relevance to the fatal alcohol syndrome

Day 10 rat embryos grown $\text{in}$$\text{vitro}$ showed significant retardation in growth and development when culture media contained acetaldehyde. A concentration-response range for acetaldehyde-induced embryotoxicity was defined, from no effect at 5μM to complete lethality at 100μM. The relative teratogenicity of ethanol and acetaldehyde, and the potential roles of these compounds in producing the Fetal Alcohol Syndrome are discussed.Despite intensive investigation into alcohol teratogenicity, the mechanism that produces the Fetal Alcohol Syndrome (FAS) remains unknown. Observed anomalies may result from direct embryonic exposure to ethanol or one of its metabolites, or from some indirect effect such as altered placental function or maternal nutritional status. Use of $\text{in}$$\text{vitro}$ techniques allows study of direct embryonic exposures in the absence of indirect influences. Under such conditions, ethanol has been found to exert direct embryotoxicity (1). Rat embryos, grown as cultured explants and subjected to ethanol concentrations of 32.5 or 65mM, were retarded in growth and development when compared to untreated controls. In this paper, we report direct embryotoxic effects of acetaldehyde, the primary metabolite of ethanol, at concentrations as low as 25μM.Acetaldehyde teratogenicity has not been extensively studied. Veghelyi et al. (2) and Lambert, Papp and Nishiura (3) employed a combination of ethanol and disulfiram (an inhibitor of acetaldehyde-oxidizing enzymes). Teratogenic effects exceeded expectations based upon assumption of an additive interaction between these two compounds, and were attributed to elevated maternal blood acetaldehyde. O'Shea and Kauffman (4,5) and Dreosti et al. (6) administered acetaldehyde to pregnant animals by injection. Treatment resulted in retarded growth and development, decreased DNA synthesis, and increased frequencies of malformation and resorption. While these studies imply a role for acetaldehyde in alcohol-induced teratogenesis, indirect effects through altered maternal or placental factors cannot be eliminated. We present here the first concentration-response data for direct ebryonic exposure to acetaldehyde.     

Oxygen and temperature effects on acetaldehyde-induced catabolite inactivation in Pichia pastoris

Summary Exposure of methylotrophic yeasts to other carbon sources after growth on methanol results in catabolite inactivation. As a result, peroxisomes are rapidly degraded effectively disabling the metabolic pathway initiated by alcohol oxidase in favour of a more energetically favourable route. A model equation has been developed to describe the effect of temperature, dissolved oxygen concentration and acetaldehyde (catabolite) concentration on catabolite inactivation in Pichia pastoris. When pre-exposed to 4 g/l acetaldehyde at 30°C, the rate of conversion of ethanol to acetaldehyde decreased by 75%. Inactivation was reduced to 45% at 30°C by reducing the dissolved oxygen concentration. At high dissolved oxygen concentration, enzyme function was only inactivated by 20% if the temperature during the period of exposure to acetaldehyde was reduced to 5°C. The influence of acetaldehyde can be eliminated completely by operating at 5°C and low dissolved oxygen concentrations. Application of these findings to process design has enabled us to conduct preliminary reactions in laboratory-scale reactors that have yielded acetaldehyde concentrations greater than 3 M (130 g/l) in 4 h. Offsprint requests to: S. J. B. Duff     

Chemiluminescence by Listeria monocytogenes.

Listeria monocytogenes cells suspended in brain heart infusion broth or in carbonated saline solution emitted light (chemiluminescence) that could be detected by a liquid scintillation spectrometer. This chemiluminescence was inhibited by superoxide dismutase and catalase but not by the hydroxyl radical scavengers mannitol and benzoate; it was also dependent upon and proportional to the carbonate ion concentration in the medium. Organisms suspended in carbonated saline solution which had ceased to chemiluminesce immediately began to chemiluminesce again when acetaldehyde was added but not when glucose, sucrose, or xanthine was added. Acetaldehyde-induced chemiluminescence was inhibited by suproxide dismutase and catalase but not by allopurinol. Our data indicate that the superoxide anion, hydrogen peroxide, and the carbonate ion are involved in chemiluminescence by L. monocytogenes. Chemiluminescence is apparently initiated by the extracellular generation of superoxide anon by this organism. The mechanism for the production of the superoxide anion is not known, but xanthine oxidase does not appear to be involved.     

A biochemically structured model for Saccharomyces cerevisiae.

A biochemically structured model for the aerobic growth of Saccharomyces cerevisiae on glucose and ethanol is presented. The model focuses on the pyruvate and acetaldehyde branch points where overflow metabolism occurs when the growth changes from oxidative to oxido-reductive. The model is designed to describe the onset of aerobic alcoholic fermentation during steady-state as well as under dynamical conditions, by triggering an increase in the glycolytic flux using a key signalling component which is assumed to be closely related to acetaldehyde. An investigation of the modelled process dynamics in a continuous cultivation revealed multiple steady states in a region of dilution rates around the transition between oxidative and oxido-reductive growth. A bifurcation analysis using the two external variables, the dilution rate, D, and the inlet concentration of glucose, S(f), as parameters, showed that a fold bifurcation occurs close to the critical dilution rate resulting in multiple steady-states. The region of dilution rates within which multiple steady states may occur depends strongly on the substrate feed concentration. Consequently a single steady state may prevail at low feed concentrations, whereas multiple steady states may occur over a relatively wide range of dilution rates at higher feed concentrations.     

Peroxisomal dihydroxyacetone phosphate acyltransferase. Effect of acetaldehyde on the intact and solubilized activity

The peroxisomal enzyme dihydroxyacetone phosphate (DHAP) acyltransferase shows a differential response to acetaldehyde. Employing whole peroxisomes, the enzyme displays a 130-400% stimulation of activity when assayed in the presence of 10-250 mM acetaldehyde. Following taurocholate solubilization of the enzyme the response to 0.25 M acetaldehyde is one of almost total inhibition. This inhibition of the taurocholate-solubilized enzyme is not observed at acetaldehyde concentrations below 200 mM. The stimulation of DHAP acyltransferase by acetaldehyde is solely a response of the peroxisomal enzyme as evidenced by its insensitivity to N-ethylmaleimide and 5 mM glycerol 3-phosphate. Furthermore, microsomal dihydroxyacetone phosphate acyltransferase activity is inhibited at all acetaldehyde concentrations. The activation of membrane-bound DHAP acyltransferase by acetaldehyde appears to be specific for this enzyme in comparison to several other peroxisomal and microsomal enzymes. The specificity of activation and differential response of the peroxisomal enzyme to acetaldehyde indicates that the microenvironment of the peroxisomal membrane is important for normal enzymatic function of this enzyme.     

Effect of acetaldehyde on Saccharomyces cerevisiae and Zymomonas mobilis subjected to environmental shocks

The lag phase of Saccharomyces cerevisiae subjected to a step increase in temperature or ethanol concentration was reduced by as much as 60% when acetaldehyde was added to the medium at concentrations less than 0.1 g/L. Maximum specific growth rates were also substantially increased. Even greater proportional reductions in lag time due to acetaldehyde addition were observed for ethanol-shocked cultures of Zymomonas mobilis. Acetaldehyde had no effect on S. cerevisiae cultures started from stationary phase inocula in the absence of environmental shock and its lag-reducing effects were greater in complex medium than in a defined synthetic medium. Acetaldehyde reacted strongly with the ingredients of complex culture media. It is proposed that the effect of added acetaldehyde may be to compensate for the inability of cells to maintain transmembrane acetaldehyde gradients following an environmental shock. (c) 1997 John Wiley & Sons, Inc.     

Covalent binding of acetaldehyde to type III collagen

Incubation of neutral salt soluble type III pN-collagen with [14C]acetaldehyde in vitro resulted in the formation of spontaneously stable acetaldehyde-protein adducts. This reaction occurred primarily at lysine residues and it was not affected by 0.2-2 mM concentrations of ascorbate but addition of sodiumcyanoborohydride increased the stable adducts by 3-5-fold. When confluent cultures of human skin fibroblasts were incubated with physiologically relevant concentrations of acetaldehyde, it became covalently bound to type III procollagen secreted into the medium. We propose that acetaldehyde binding to collagen fibrils occurs in vivo following chronic alcohol consumption.     

Acetaldehyde addition and pre-adaptation to the stressor together virtually eliminate the ethanol-induced lag phase in Saccharomyces cerevisiae

AIMS: To show that the ethanol-induced lag phase in yeast can be almost eliminated by combining pre-adaptation with acetaldehyde supplementation. METHODS AND RESULTS: Pre-adaptation to noninhibitory concentrations of ethanol and supplementation of unadapted cultures with acetaldehyde each separately reduced the lag phase of ethanol-inhibited cultures by c. 70%. By combining the two methods the ethanol-induced lag phase was virtually eliminated (90% reduction in lag time). CONCLUSIONS: Pre-adaptation to ethanol and acetaldehyde supplementation appear to promote yeast growth through different mechanisms, which are additive when combined. SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of the above procedures is a potentially powerful tool for reducing the lag of stressed cultures, which may have practical applications: e.g. in reducing the lag of yeasts inoculated into lignocellulosic hydrolysates employed in fuel ethanol production.     

Autoconditioning factor relieves ethanol-induced growth inhibition of Saccharomyces cerevisiae.

Viable Saccharomyces cerevisiae suspended in medium containing growth-inhibiting concentrations of ethanol produce a metabolite that relieves growth inhibition. This autoconditioning of the medium by yeasts is due to the formation of small amounts (0.01%, vol/vol) of acetaldehyde. The effect is duplicated precisely in fresh medium by the addition of acetaldehyde. Acetaldehyde does not increase the yield of or accelerate ethanol production by the organism. Ethanol-induced modifications of membrane order in the plasma membranes, as measured by steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene, were not resolved by exogenously added acetaldehyde.     

Autoconditioning factor relieves ethanol-induced growth inhibition of Saccharomyces cerevisiae

Viable Saccharomyces cerevisiae suspended in medium containing growth-inhibiting concentrations of ethanol produce a metabolite that relieves growth inhibition. This autoconditioning of the medium by yeasts is due to the formation of small amounts (0.01%, vol/vol) of acetaldehyde. The effect is duplicated precisely in fresh medium by the addition of acetaldehyde. Acetaldehyde does not increase the yield of or accelerate ethanol production by the organism. Ethanol-induced modifications of membrane order in the plasma membranes, as measured by steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene, were not resolved by exogenously added acetaldehyde.     

Participation of acetaldehyde dehydrogenases in ethanol and pyruvate metabolism of the yeast Saccharomyces cerevisiae.

This work was undertaken to clarify the role of acetaldehyde dehydrogenases in Saccharomyces cerevisiae metabolism during growth on respiratory substrates. Until now, there has been little agreement concerning the ability of mutants deleted in gene ALD4, encoding mitochondrial acetaldehyde dehydrogenase, to grow on ethanol. Therefore we constructed mutants in two parental strains (YPH499 and W303-1a). Some differences appeared in the growth characteristics of mutants obtained from these two parental strains. For these experiments we used ethanol, pyruvate or lactate as substrates. Mitochondria can oxidize lactate into pyruvate using an ATP synthesis-coupled pathway. The ald4Delta mutant derived from the YPH499 strain failed to grow on ethanol, but growth was possible for the ald4Delta mutant derived from the W303-1a strain. The co-disruption of ALD4 and PDA1 (encoding subunit E1alpha of pyruvate dehydrogenase) prevented the growth on pyruvate for both strains but prevented growth on lactate only in the double mutant derived from the YPH499 strain, indicating that the mutation effects are strain-dependent. To understand these differences, we measured the enzyme content of these different strains. We found the following: (a) the activity of cytosolic acetaldehyde dehydrogenase in YPH499 was relatively low compared to the W303-1a strain; (b) it was possible to restore the growth of the mutant derived from YPH499 either by addition of acetate in the media or by introduction into this mutant of a multicopy plasmid carrying the ALD6 gene encoding cytosolic acetaldehyde dehydrogenase. Therefore, the lack of growth of the mutant derived from the YPH499 strain seemed to be related to the low activity of acetaldehyde oxidation. Therefore, when cultured on ethanol, the cytosolic acetaldehyde dehydrogenase can partially compensate for the lack of mitochondrial acetaldehyde dehydrogenase only when the activity of the cytosolic enzyme is sufficient. However, when cultured on pyruvate and in the absence of pyruvate dehydrogenase, the cytosolic acetaldehyde dehydrogenase cannot compensate for the lack of the mitochondrial enzyme because the mitochondrial form produces intramitochondrial NADH and consequently ATP through oxidative phosphorylation.     

Determination of metformin hydrochloride using precolumn derivatization with acetaldehyde and capillary electrophoresis coupled with electrochemiluminescence

A novel method was developed using capillary electrophoresis (CE) coupled with tris(2,2′‐bipyridyl)ruthenium(II) electrogenerated chemiluminescence (ECL) for highly sensitive detection of metformin hydrochloride (MH) derivatizatized with acetaldehyde. The precolumn derivatization of MH with acetaldehyde was performed in phosphate buffer solution (0.3 mol/L, pH 7.5) at room temperature for 120 min. The effects of acetaldehyde concentration, buffer pH, electrokinetic voltage and injection time were investigated. Under optimized detection conditions, the MH ECL detection sensitivity was more than 120 times that without derivatization. The linear concentration range for MH was 0.001–15.00 μg/mL (with a correlation coefficient of 0.9992). The detection limit was 0.31 ng/mL with a signal:noise ratio of 3. The recoveries of MH in human urine were in the range 98.50–99.72%. Copyright © 2011 John Wiley & Sons, Ltd.     

Acetaldehyde inhibits the yeast-to-hypha conversion and biofilm formation in <Emphasis Type="Italic">Candida albicans</Emphasis>

In Candida albicans, alcohol metabolism is implicated in biofilm formation. The alcohol dehydrogenase gene (ADH1) is involved in the conversion of acetaldehyde to ethanol and reported to be downregulated during biofilm formation. C. albicans produces acetaldehyde under both in vivo and in vitro conditions. Mutations in ADH genes result in increased acetaldehyde production in vitro, but studies are lacking on the morphogenetic role(s) of acetaldehyde in C. albicans. We report here that acetaldehyde at a concentration of 7 mM was able to inhibit the conversion from yeast to hyphal forms induced by four standard inducers at 37°C. The hyphal inhibitory concentrations did not adversely affect the growth and viability of C. albicans cells. The same concentration of acetaldehyde also significantly inhibited biofilm development, and only adhered yeast cells were found. We hypothesize that acetaldehyde produced by C. albicans may exert a morphogenetic regulatory role influencing yeast-to-hypha conversion, biofilm formation, dissemination and establishment of infection.