Effect of acetaldehyde and cyanamide on the metabolism of formaldehyde by hepatocytes, mitochondria, and soluble supernatant from rat liver

Formaldehyde can be metabolized primarily by two different pathways, one involving oxidation by the low-Km mitochondrial aldehyde dehydrogenase, the other involving a specific, glutathione-dependent, formaldehyde dehydrogenase. To estimate the roles played by each enzyme in formaldehyde metabolism by rat hepatocytes, experiments with acetaldehyde and cyanamide, a potent inhibitor of the low-Km aldehyde dehydrogenase were carried out. The glutathione-dependent oxidation of formaldehyde by 100,000g rat liver supernatant fractions was not affected by either acetaldehyde or by cyanamide. By contrast, the uptake of formaldehyde by intact mitochondria was inhibited 75 to 90% by cyanamide. Acetaldehyde inhibited the uptake of formaldehyde by mitochondria in a competitive fashion. Formaldehyde was a weak inhibitor of the oxidation of acetaldehyde by mitochondria, suggesting that, relative to formaldehyde, acetaldehyde was a preferred substrate. In isolated hepatocytes, cyanamide, which inhibited the oxidation of acetaldehyde by 75 to 90%, produced only 30 to 50% inhibition of formaldehyde uptake by cells as well as of the production of 14CO2 and of formate from [14C]formaldehyde. The extent of inhibition by cyanamide was the same as that produced by acetaldehyde (30-40%). In the presence of cyanamide, acetaldehyde was no longer inhibitory, suggesting that acetaldehyde and cyanamide may act at the same site(s) and inhibit the same formaldehyde-oxidizing enzyme system. These results suggest that, in rat hepatocytes, formaldehyde is oxidized by cyanamide- and acetaldehyde-sensitive (low-Km aldehyde dehydrogenase) and insensitive (formaldehyde dehydrogenase) reactions, and that both enzymes appear to contribute about equally toward the overall metabolism of formaldehyde.     

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.     

Inhibition of mitochondrial aldehyde dehydrogenase and acetaldehyde oxidation by the glutathione-depleting agents diethylmaleate and phorone

Experiments were carried out to study the effect of two commonly used glutathione-depleting agents, diethylmaleate and phorone, on the oxidation of acetaldehyde and the activity of aldehyde dehydrogenase. The oxidation of acetaldehyde by intact hepatocytes was inhibited when the cells were incubated with diethylmaleate. Washing and resuspending the cells in diethylmaleate-free medium afforded protection against the inhibition of acetaldehyde oxidation. The oxidation of acetaldehyde by isolated rat liver mitochondria as well as by disrupted mitochondria in the presence of excess NAD+ was inhibited by diethylmaleate or phorone, indicating inhibition of the low-Km aldehyde dehydrogenase. In addition, diethylmaleate inhibited oxidation of acetaldehyde by the high-Km cytosolic aldehyde dehydrogenase. Significant accumulation of acetaldehyde occurred when ethanol was oxidized by hepatocytes in the presence, but not in the absence, of diethylmaleate. Thus, diethylmaleate blocks the oxidation of added or metabolically generated acetaldehyde, analogous to results with other inhibitors of the low-Km aldehyde dehydrogenase such as cyanamide. These results suggest that caution should be used in interpreting the effects of diethylmaleate or phorone on metabolic reactions, especially those involving metabolism of aldehydes such as formaldehyde, because, in addition to depleting glutathione, these agents inhibit the low-Km aldehyde dehydrogenase.     

Inhibition of the oxidation of acetaldehyde and formaldehyde by hepatocytes and mitochondria by crotonaldehyde

Crotonaldehyde was oxidized by disrupted rat liver mitochondrial fractions or by intact mitochondria at rates that were only 10 to 15% that of acetaldehyde. Although a poor substrate for oxidation, crotonaldehyde is an effective inhibitor of the oxidation of acetaldehyde by mitochondrial aldehyde dehydrogenase, by intact mitochondria, and by isolated hepatocytes. Inhibition by crotonaldehyde was competitive with respect to acetaldehyde, and the Ki for crotonaldehyde was about 5 to 20 microM. Crotonaldehyde had no effect on the oxidation of glutamate or succinate. Very low levels of acetaldehyde were detected during the metabolism of ethanol. Crotonaldehyde increased the accumulation of acetaldehyde more than 10-fold, indicating that crotonaldehyde, besides inhibiting the oxidation of added acetaldehyde, also inhibited the oxidation of acetaldehyde generated by the metabolism of ethanol. Formaldehyde was a substrate for the low-Km mitochondrial aldehyde dehydrogenase, as well as for a cytosolic, glutathione-dependent formaldehyde dehydrogenase. Crotonaldehyde was a potent inhibitor of mitochondrial oxidation of formaldehyde, but had no effect on the activity of formaldehyde dehydrogenase. In hepatocytes, crotonaldehyde produced about 30 to 40% inhibition of formaldehyde oxidation, which was similar to the inhibition produced by cyanamide. This suggested that part of the formaldehyde oxidation occurred via the mitochondrial aldehyde dehydrogenase, and part via formaldehyde dehydrogenase. The fact that inhibition by crotonaldehyde is competitive may be of value since other commonly used inhibitors of aldehyde dehydrogenase are irreversible inhibitors of the enzyme.     

Inhibition of hepatic aldehyde dehydrogenases in the rat by calcium carbimide (calcium cyanamide)

Oral administration of 7.0 mg/kg calcium carbimide (calcium cyanamide, CC) to the rat produced differential inhibition of hepatic aldehyde dehydrogenase (ALDH) isozymes, as indicated by the time-course profiles of enzyme activity. The low-Km mitochondrial ALDH was most susceptible to inhibition following CC administration, with complete inhibition occurring at 0.5 h and return to control activity at 96 h. The low-Km cytosolic and high-Km mitochondrial, cytosolic, and microsomal ALDH isozymes were inhibited to a lesser degree and (or) for a shorter duration compared with the mitochondrial low-Km enzyme. The time course of carbimide, the hydrolytic product of CC, was determined in plasma following oral administration of 7.0 mg/kg CC to the rat. The maximum plasma carbimide concentration (102 ng/mL) occurred at 1 h and the apparent elimination half-life in plasma was 1.5 h. Carbimide was not measurable in the liver during the 6.5 h time interval when carbimide was present in the plasma. There were negative, linear correlations between plasma carbimide concentration and hepatic low-Km mitochondrial, low-Km cytosolic, and high-Km microsomal ALDH activities. In vitro studies demonstrated that carbimide, at concentrations obtained in plasma following oral CC administration, produced only 19% inhibition of low-Km mitochondrial ALDH and no inhibition of low-Km cytosolic and high-Km microsomal ALDH isozymes. These data demonstrate that carbimide, itself, is not primarily responsible for hepatic ALDH inhibition in vivo following oral CC administration. It would appear that carbimide must undergo metabolic conversion in vivo to inhibit hepatic ALDH enzymes, which is supported by the observation of no measurable carbimide in the liver when ALDH was maximally inhibited following oral CC administration.     

Aldehyde dehydrogenase activity as the rate-limiting factor for acetaldehyde metabolism in rat liver

The velocity of acetaldehyde metabolism in rat liver may be governed either by the rate of regeneration of NAD from NADH through the electron transport system or by the activity of aldehyde dehydrogenase (ALDH). Measurements of oxygen consumption revealed that the electron transport system was capable of reoxidizing ALDH-generated NADH much faster than it was produced and hence was not rate-limiting for aldehyde metabolism. To confirm that ALDH activity was the rate-limiting factor, low-Km ALDH in slices or intact mitochondria was partially inhibited by treatment with cyanamide and the rate of acetaldehyde metabolism measured. Any inhibition of low-Km ALDH resulted in a decreased rate of acetaldehyde metabolism, indicating that no excess of low-Km ALDH existed. Approximately 40% of the metabolism of 200 microM acetaldehyde in slices was not catalyzed by low-Km ALDH. Fifteen of this 40% was catalyzed by high-Km ALDH. A possible contribution by aldehyde oxidase was ruled out through the use of a competitive inhibitor, quinacrine. Acetaldehyde binding to cytosolic proteins may account for the remainder. By measuring acetaldehyde accumulation during ethanol metabolism, it was also established that low-Km ALDH activity was rate-limiting for acetaldehyde oxidation during concomitant ethanol oxidation.     

Dye-linked dehydrogenase activities for formate and formate esters in Amycolatopsis methanolica. Characterization of a molybdoprotein enzyme active with formate esters and aldehydes.

Cell-free extracts of methanol-grown Amycolatopsis methanolica contain dye-linked dehydrogenase activities for formate and methyl formate. Fractionation of the extracts revealed that the (unstable) activity for formate resides in membrane particles, while that for methyl formate belongs to a soluble enzyme that was purified and characterized. The enzyme, indicated as formate-ester dehydrogenase, appeared to be a molybdoprotein (4 Fe, 3 or 4 S, 1 Mo and 1 FAD were found for each enzyme molecule), with a molecular mass of 186 kDa and consisting of two subunits of equal size. Product identification suggests that the formate moiety in the ester becomes hydroxylated to a carbonate group after which the unstable alkyl carbonate decomposes into CO2 and the alcohol moiety. Based on structural and catalytic characteristics, the enzyme appears to be very similar to an enzyme isolated from Comamonas testosteroni [Poels, P. A., Groen, B. W. & Duine, J. A. (1987) Eur. J. Biochem. 166, 575-579] which was at that time considered to be an aldehyde dehydrogenase. Formate-ester dehydrogenase activity appeared to be present in several other bacteria. Possible roles for the A. methanolica enzyme in C1 dissimilation (oxidation of methyl formate to methanol and CO2 or a factor-formate adduct to factor plus CO2) or in general aldehyde oxidation, are discussed.     

Thiols in formaldehyde dissimilation and detoxification

Glutathione is not a universal coenzyme for formaldehyde oxidation. MySH (mycothiol, 1-O-(2'-[N-acetyl-L-cysteinyl]amido-2'-deoxy-alpha-D-glucopyranosyl)-D-m yo-inositol) is GSH's counterpart as coenzyme in formaldehyde dehydrogenase from certain gram-positive bacteria. However, formaldehyde dissimilation and detoxification not only proceed via thiol-dependent but also via thiol-independent dehydrogenases. The distinct structures and enzymatic properties of MySH-dependent and GSH-dependent formaldehyde dehydrogenases could provide clues for development of selective drugs against pathogenic Mycobacteria. It is to be expected that other new types of thiol-dependent formaldehyde dehydrogenases will be discovered in the future. Indications exist that the product of thiol-dependent formaldehyde oxidation, the thiol formate ester, is not only hydrolytically converted into thiol and formate but can also be oxidatively converted in some cases by a molybdoprotein aldehyde dehydrogenase into the corresponding carbonate ester, decomposing spontaneously into CO2 and the thiol.     

Oxidation of C1-compounds in Pseudomonas C.

Extracts of Pseudomonas C grown on methanol as a sole carbon and energy source contain a methanol dehydrogenase activity which can be coupled to phenazine methosulfate. This enzyme catalyzes two reactions namely the conversion of methanol to formaldehyde (phenazine methosulfate coupled) and the oxidation of formaldehyde to formate (2,6-dichloroindophenol-coupled). Activities of glutathione-dependent formaldehyde dehydrogenase (NAD+) and formate dehydrogenase (NAD+) were also detected in the extracts. The addition of D-ribulose 5-phosphate to the reaction mixtures caused a marked increase in the formaldehyde-dependent reduction of NAD+ or NADP+. In addition, the oxidation of [14C]formaldehyde to CO2, by extracts of Pseudomonas C, increased when D-ribulose 5-phosphate was present in the assay mixtures. The amount of radioactivity found in CO2, was 6;8-times higher when extracts of methanol-grown Pseudomonas C were incubated for a short period of time with [1-14C]glucose 6-phosphate than with [U-14C]glucose 6-phosphate. These data, and the presence of high specific activities of hexulose phosphate synthase, phosphoglucoisomerase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase indicate that in methanol-grown Pseudomonas C, formaldehyde carbon is oxidized to CO2 both via a cyclic pathway which includes the enzymes mentioned and via formate as an oxidation intermediate, with the former predominant.     

Purification and properties of formaldehyde dehydrogenase and formate dehydrogenase from Candida boidinii.

Formaldehyde hydrogenase and formate dehydrogenase were purified 130-fold and 19-fold respectively from Candida boidinii grown on methanol. The final enzyme preparations were homogenous as judged by acrylamide gel electrophoresis and by sedimentation in an ultracentrifuge. The molecular weights of the enzymes were determined by sedimentation equilibrium studies and calculated as 80000 and 74000 respectively. Dissociation into subunits was observed by treatment with sodium dodecylsulfate. The molecular weights of the polypeptide chains were estimated to be 40000 and 36000 respectively. The NAD-linked formaldehyde dehydrogenase specifically requires reduced glutathione for activity. Besides formaldehyde only methylglyoxal served as a substrate but no other aldehyde tested. The Km values were found to be 0.25 mM for formaldehyde, 1.2 mM for methylglyoxal, 0.09 mM for NAD and 0.13 mM for glutathione. Evidence is presented which demonstrates that the reaction product of the formaldehyde-dehydrogenase-catalyzed oxidation of formaldehyde is S-formylglutathione rather than formate. The NAD-linked formate dehydrogenase catalyzes specifically the oxidation of formate to carbon dioxide. The Km values were found to be 13 mM for formate and 0.09 mM for NAD.     

Cofactor-dependent pathways of formaldehyde oxidation in methylotrophic bacteria

Methylotrophic bacteria can grow on a number of substrates as energy source with only one carbon atom, such as methanol, methane, methylamine, and dichloromethane. These compounds are metabolized via the cytotoxin formaldehyde. The formaldehyde consumption pathways, especially the pathways for the oxidation of formaldehyde to CO(2) for energy metabolism, are a central and critical part of the metabolism of these aerobic bacteria. Principally, two main types of pathways for the conversion of formaldehyde to CO(2) have been described: (1) a cyclic pathway initiated by the condensation of formaldehyde with ribulose monophosphate, and (2) distinct linear pathways that involve a dye-linked formaldehyde dehydrogenase or C(1) unit conversion bound to the cofactors tetrahydrofolate (H(4)F), tetrahydromethanopterin (H(4)MPT), glutathione (GSH), or mycothiol (MySH). The pathways involving the four cofactors have in common the following sequence of events: the spontaneous or enzyme-catalyzed condensation of formaldehyde and the respective C(1) carrier, the oxidation of the cofactor-bound C(1) unit and its conversion to formate, and the oxidation of formate to CO(2). However, the H(4)MPT pathway is more complex and involves intermediates that were previously known solely from the energy metabolism of methanogenic archaea. The occurrence of the different formaldehyde oxidation pathways is not uniform among different methylotrophic bacteria. The pathways are in part also used by other organisms to provide C(1) units for biosynthetic reactions (e.g., H(4)F-dependent enzymes) or detoxification of formaldehyde (e.g., GSH-dependent enzymes).     

Biochemical properties of rat liver mitochondrial aldehyde dehydrogenase with respect to oxidation of formaldehyde.

The oxidation of formaldehyde by rat liver mitochondria in the presence of 50 mM phosphate was enhanced 2-fold by exogenous NAD+. Absolute requirement of NAD+ for formaldehyde oxidation was demonstrated by depleting the mitochondria of their NAD+ content (4.6 nmol/mg of protein), followed by reincorporation of the NAD+ into the depleted mitochondria. Aldehyde (formaldehyde) dehydrogenase activity was completely abolished in the depleted mitochondria, but the enzyme activity was restored to control levels following reincorporation of the pyridine nucleotide. Phosphate stimulation of formaldehyde oxidation could not be explained fully by the phosphate-induced swelling which enhances membrane permeability to NAD+, since stimulation of the enzyme activity by increased phosphate concentrations was still observed in the absence of exogenous NAD+. The Km for formaldehyde oxidation by the mitochondria was found to be 0.38 nM, a value similar to that obtained with varying concentrations of NAD+; both Vmax values were very similar, giving a value of 70 to 80 nmol/min/mg of protein. The pH optimum for the mitochondrial enzyme was 8.0. Inhibition of the enzyme activity by anaerobiosis was apparently due to the inability of the respiratory chain to oxidize the generated NADH. The inhibition of mitochondrial formaldehyde oxidation by succinate was found to be due to a lowering of the NAD+ level in the mitochondria. Succinate also inhibited acetaldehyde oxidation by the mitochondria. Malonate, a competitive inhibitor of succinic dehydrogenase, blocked the inhibitory effect of succinate. The respiratory chain inhibitors, rotenone, and antimycin A plus succinate, strongly inhibited formaldehyde oxidation by apparently the same mechanism, although the crude enzyme preparation (freed from the membrane) was slightly sensitive to rotenone. The mitochondria were subfractionated, and 85% of the enzyme activity was found in the inner membrane fraction (mitoplast). Furthermore, separation into inner membrane and matrix components indicated a distribution of aldehyde dehydrogenase activity similar to malic dehydrogenase.     

Chemically-induced glutathione depletion and lipid peroxidation

Malondialdehyde (MDA) formation in mouse liver homogenates was measured in the presence of various glutathione depletors (5 mmol/l). After a lag phase of 90 min, the MDA formation increased from 1.25 nmol/mg protein to 14.5 nmol/mg in the presence of diethyl maleate (DEM), to 10.5 with diethyl fumarate (DEF) and to 4 with cyclohexenon by 150 min. It remained at 1.25 nmol/mg with phorone and in the control. On the other hand, glutathione (GSH) dropped from 55 nmol/mg to 50 nmol/mg in the control to, < 1 with DEM, to 46 with DEF, to 3 with cyclohexenon and to 7 with phorone. The data show that the potency to deplete GSH is not related to MDA production in this system. DEM stimulated in vitro ethane evolution in a concentration-dependent manner and was strongly inhibited by SKF 525A. From type I binding spectra to microsomal pigments the following spectroscopic binding constants were determined: 2.5 mmol/l for phorone, 1.2 mmol/l for cyclohexenon, 0.5 mmol/l for DEM and 0.3 mmol/l for DEF. In isolated mouse liver microsomes NADPH-cytochrome P-450 reductase and NADH-cytochrome b₅ reductase activity were unaffected by the presence of DEM, whereas ethoxycoumarin dealkylation was inhibited. Following in vivo pretreatment, hepatic microsomal electron flow as determined in vitro was augmented in the presence of depleting as well as non-depleting agents, accompanied by a shift from O₂⁻ to H₂O₂ production. It is concluded that it is not the absence of GSH which causes lipid peroxidation after chemically-induced GSH depletion but rather the interaction of the chemicals with the microsomal monoxygenase system.     

Glutathione participation in the regulation of methanol metabolism in yeasts

The activity of enzymes involved in methanol oxidation and assimilation as well as the levels of formaldehyde and glutathione were determined during batch cultivation of Candida boidinii KD1 in a medium with methanol. The distribution of [14C]methanol between oxidative and biosynthetic processes in the yeast was analysed. Changes in the concentrations of formaldehyde and glutathione were found to correlate with the activity of formaldehyde dehydrogenase. The results indicate that an increase in the concentration of reduced glutathione (GSH) at the early logarithmic phase of the yeast growth stimulates formaldehyde oxidation via formate to carbon dioxide whereas a subsequent decrease in the concentration of GSH favours formaldehyde assimilation.     

The formaldehyde metabolic detoxification enzyme systems and molecular cytotoxic mechanism in isolated rat hepatocytes

The toxicity and carcinogenicity of formaldehyde (HCHO) has been attributed to its ability to form adducts with DNA and proteins. A marked decrease in mitochondrial membrane potential and inhibition of mitochondrial respiration that was accompanied by reactive oxygen species formation occurred when isolated rat hepatocytes were incubated with low concentrations of HCHO in a dose-dependent manner. Hepatocyte GSH was also depleted by HCHO in a dose-dependent manner. At higher HCHO concentrations, lipid peroxidation ensued followed by cell death. Cytotoxicity studies were conducted in which isolated hepatocytes exposed to HCHO were treated with inhibitors of HCHO metabolising enzymes. There was a marked increase in HCHO cytotoxicity when either alcohol dehydrogenase or aldehyde dehydrogenase was inhibited. Inhibition of GSH-dependent HCHO dehydrogenase activity by prior depletion of GSH markedly increased hepatocyte susceptibility to HCHO. In each case, cytotoxicity was dose-dependent and corresponded with a decrease in hepatocyte HCHO metabolism and increased lipid peroxidation. Antioxidants and iron chelators protected against HCHO cytotoxicity. Cytotoxicity was also prevented, when cyclosporine or carnitine was added to prevent the opening of the mitochondrial permeability transition pore which further suggests that HCHO targets the mitochondria. Thus, HCHO-metabolising gene polymorphisms would be expected to have toxicological consequences on an individual's susceptibility to HCHO toxicity and carcinogenesis.     

The formaldehyde metabolic detoxification enzyme systems and molecular cytotoxic mechanism in isolated rat hepatocytes

The toxicity and carcinogenicity of formaldehyde (HCHO) has been attributed to its ability to form adducts with DNA and proteins. A marked decrease in mitochondrial membrane potential and inhibition of mitochondrial respiration that was accompanied by reactive oxygen species formation occurred when isolated rat hepatocytes were incubated with low concentrations of HCHO in a dose-dependent manner. Hepatocyte GSH was also depleted by HCHO in a dose-dependent manner. At higher HCHO concentrations, lipid peroxidation ensued followed by cell death. Cytotoxicity studies were conducted in which isolated hepatocytes exposed to HCHO were treated with inhibitors of HCHO metabolising enzymes. There was a marked increase in HCHO cytotoxicity when either alcohol dehydrogenase or aldehyde dehydrogenase was inhibited. Inhibition of GSH-dependent HCHO dehydrogenase activity by prior depletion of GSH markedly increased hepatocyte susceptibility to HCHO. In each case, cytotoxicity was dose-dependent and corresponded with a decrease in hepatocyte HCHO metabolism and increased lipid peroxidation. Antioxidants and iron chelators protected against HCHO cytotoxicity. Cytotoxicity was also prevented, when cyclosporine or carnitine was added to prevent the opening of the mitochondrial permeability transition pore which further suggests that HCHO targets the mitochondria. Thus, HCHO-metabolising gene polymorphisms would be expected to have toxicological consequences on an individual's susceptibility to HCHO toxicity and carcinogenesis.     

Glutathione participates in the modulation of starvation-induced autophagy in carcinoma cells

Glutathione (γ-L-glutamyl-L-cysteinyl-glycine, GSH) is the most abundant low molecular weight, thiol-containing compound within the cells and has a primary role in the antioxidant defense and intracellular signaling. Here we demonstrated that nutrient deprivation led to a significant decrease of intracellular GSH levels in three different carcinoma cell lines. This phenomenon was dependent on ABCC1-mediated GSH extrusion, along with GCL inhibition and, to a minor extent, the formation of GSH-protein mixed disulfides that synergistically contributed to the modulation of autophagy by shifting the intracellular redox state toward more oxidizing conditions. Modulation of intracellular GSH by inhibiting its de novo synthesis through incubation with buthionine sulfoximine, or by maintaining its levels through GSH ethyl ester, affected the oxidation of protein thiols, such as PRDXs and consequently the kinetics of autophagy activation. We also demonstrated that thiol-oxidizing or -alkylating agents, such as diamide and diethyl maleate activated autophagy, corroborating the evidence that changes in thiol redox state contributed to the occurrence of autophagy.     

Hepatic organelle interaction. IV. Mechanism of succinate enhancement of formaldehyde accumulation from endoplasmic reticulum N-dealkylations

Further evidence for organelle interaction during drug metabolism by the liver is presented. The apparent stimulation by succinate of formaldehyde accumulation in the medium, which was reported to occur with liver slices and homogenates as well as with mitochondria plus microsomes, has been shown to be the result of succinate inhibition of mitochondrial aldehyde dehydrogenase. The mechanism of succinate inhibition is shown to be by reverse electron transport, and an increase in the NADH to NAD+ ratio in the mitochondria; the aldehyde dehydrogenase requires the oxidized form of the pyridine nucleotide as its cofactor. Studies on in vitro N-demethylation by liver microsomes and endoplasmic reticulum segments which cosediment with the mitochondria indicate that formaldehyde produced by the mixed function oxidase is handled differently from formaldehyde added to the medium. The latter is mainly retained in the medium containing 5 mM semicarbazide, while the generated formaldehyde is more than 50% consumed by the mitochondria. Electron microscopy has indicated that the microsomes and the endoplasmic reticulum fragments have a tendency to align themselves close to the mitochondria when present in the same medium. Consequently, it is possible that formaldehyde released to the medium adjacent to the mitochondria, as by N-demethylation, would be exposed to semicarbazide for shorter periods than that added directly to the medium. In agreement with this suggestion, complexing of formaldehyde with semicarbazide was observed spectroscopically not to be an extremely rapid reaction even at 37 degrees C. This is believed to be the reason for the greater extent of consumption of formaldehyde generated by the endoplasmic reticulum.     

In Vivo Kinetics of Formate Metabolism in Folate-deficient and Folate-replete Rats

It is now established that the mitochondrial production of formate is a major process in the endogenous generation of folate-linked one-carbon groups. We have developed an in vivo approach involving the constant infusion of [¹³C]formate until isotopic steady state is attained to measure the rate of endogenous formate production in rats fed on either a folate-replete or folate-deficient diet. Formate was produced at a rate of 76 μmol·h⁻¹·100 g of body weight⁻¹ in the folate-replete rats, and this was decreased by 44% in folate-deficient rats. This decreased formate production was confirmed in isolated rat liver mitochondria where formate production from serine, the principal precursor of one-carbon groups, was decreased by 85%, although formate production from sarcosine and dimethylglycine (choline metabolites) was significantly increased. We attribute this unexpected result to the demonstrated production of formaldehyde by sarcosine dehydrogenase and dimethylglycine dehydrogenase from their respective substrates in the absence of tetrahydrofolate and subsequent formation of formate by formaldehyde dehydrogenase. Comparison of formate production with the ingestion of dietary formate precursors (serine, glycine, tryptophan, histidine, methionine, and choline) showed that ∼75% of these precursors were converted to formate, indicating that formate is a significant, although underappreciated end product of choline and amino acid oxidation. Ingestion of a high protein diet did not result in increased production of formate, suggesting a regulation of the conversion of these precursors at the mitochondrial level to formate.     

Downregulation of glutaredoxin but not glutathione loss leads to mitochondrial dysfunction in female mice CNS: implications in excitotoxicity

Oxidative stress, excitotoxicity and mitochondrial dysfunction play synergistic roles in neurodegeneration. Maintenance of thiol homeostasis is important for normal mitochondrial function and dysregulation of protein thiol homeostasis by oxidative stress leads to mitochondrial dysfunction and neurodegeneration. We examined the critical roles played by the antioxidant, non-protein thiol, glutathione and related enzyme, glutaredoxin in maintaining mitochondrial function during excitotoxicity caused by beta-N-oxalyl amino-L-alanine (L-BOAA), the causative factor of neurolathyrism, a motor neuron disease involving the pyramidal system. L-BOAA causes loss of GSH and inhibition of mitochondrial complex I in lumbosacral cord of male mice through oxidation of thiol groups, while female mice are resistant. Reducing GSH levels in female mice CNS by pretreatment with diethyl maleate or L-propargyl glycine did not result in inhibition of complex I activity, unlike male mice. Further, treatment of female mice depleted of GSH with L-BOAA did not induce inhibition of complex I indicating that GSH levels were not critical for maintaining complex I activity in female mice unlike their male counterpart. Glutaredoxin, a thiol disulfide oxidoreductase helps maintain redox status of proteins and downregulation of glutaredoxin results in loss of mitochondrial complex I activity. Female mice express higher levels of glutaredoxin in certain CNS regions and downregulation of glutaredoxin using antisense oligonucleotides sensitizes them to L-BOAA toxicity seen as mitochondrial complex I loss. Ovariectomy downregulates glutaredoxin and renders female mice vulnerable to L-BOAA toxicity as evidenced by activation of AP1, loss of GSH and complex I activity indicating the important role of glutaredoxin in neuroprotection. Estrogen protects against mitochondrial dysfunction caused by excitotoxicity by maintaining cellular redox status through higher constitutive expression of glutaredoxin in the CNS. Therapeutic interventions designed to upregulate glutaredoxin may offer neuroprotection against excitotoxicity in motor neurons.