Cytotoxic molecular mechanisms and cytoprotection by enzymic metabolism or autoxidation for glyceraldehyde, hydroxypyruvate and glycolaldehyde

Previously, we showed that dietary fructose or its carbonyl metabolites, glyceraldehyde and glycolaldehyde, could be oxidized by inflammatory reactive oxygen species (ROS), products of immune cells, to form highly toxic and genotoxic products, such as glyoxal. Glycolaldehyde-caused hepatocyte protein carbonylation likely resulted from glyoxal, an autoxidation product formed by ROS. Although hepatocyte protein carbonylation by glyoxal or d-glycolaldehyde was rapid, the product was unstable. Glyceraldehyde-induced protein carbonylation was slower and was also less cytotoxic. Non-toxic concentrations of H(2)O(2) were then used to mimic inflammation and oxidative stress associated with fructose-induced non-alcoholic steatohepatitis (NASH). A slow infusion of H(2)O(2) markedly increased glyoxal, glyceraldehyde, and glycolaldehyde-induced cytotoxicity and protein carbonylation. However, it had a smaller effect on glyceraldehyde-induced protein carbonylation. The cytotoxicities of both aldehydes were increased if glutathione (GSH)-depleted hepatocytes were used, presumably because of the increased ROS formation and subsequent glyoxal-induced protein carbonylation. Catalytic amounts of Cu or Fe increased the glycolaldehyde and glyceraldehyde-induced cytotoxicity and protein carbonylation resulting from autoxidation to glyoxal. Glyceraldehyde and glycolaldehyde were also detoxified by mitochondrial aldehyde dehydrogenase (ALDH2) as ALDH2 inhibitors increased their cytotoxicity. Hydroxypyruvate has not been previously tested for toxicity and was found to be the most toxic fructose metabolite. Catalytic amounts of Cu or Fe caused hydroxypruvate autoxidation, which formed extensive ROS, glycolaldehyde and glyoxal. Iron chelators EGTA or deferoxamine inhibited cytotoxicity as well as the extensive ROS formation. The Girard assay confirmed that glyoxal was a common autoxidation product from glyceraldehyde, glycolaldehyde and hydroxypyruvate.     

Cytoprotection by almond skin extracts or catechins of hepatocyte cytotoxicity induced by hydroperoxide (oxidative stress model) versus glyoxal or methylglyoxal (carbonylation model)

Oxidative and carbonyl stress are detrimental in the pathogenesis of diabetic complications, as well as in other chronic diseases. However, this process may be decreased by dietary bioactive compounds. Almond skin is an abundant source of bioactive compounds and antioxidants, including polyphenolic flavonoids, which may contribute to the decrease in oxidative and carbonyl stress. In this study, four Almond Skin Extracts (ASEI, ASEII, ASEIII, and ASEIV) were prepared by different methods and evaluated for their antioxidant activity. The order of the polyphenol content (total μM gallic acid equivalents) of the four extracts was found to be, in decreasing order of effectiveness: ASEI > ASEIII > ASEIV > ASEII. The order of Ferric-reducing antioxidant power (FRAP, μM FeSO₄/g) value, in decreasing order was ASEI (216) > ASEIII (176) > ASEIV (89) > ASEII (85). The order of ASE effectiveness for decreasing protein carbonyation induced by the copper Fenton reaction was ASEI > ASEIV > ASEII > ASEIII. The order of antioxidant effectiveness for inhibiting tertiary-butyl hydroperoxide (TBH) induced microsomal lipid peroxidation was ASEI > ASEIV > ASEII, ASEIII. Also, the order of ASE effectiveness for inhibiting TBH induced hepatocyte cell death was: ASEIII, ASEIV > ASEI, ASEII. Catechin also protected hepatocytes from TBH induced hepatocyte, lipid peroxidation and cytotoxicity. In a cell free model, equimolar concentrations of catechin or epicatechin rescued serum albumin from protein carbonylation induced by methylglyoxal (MGO). Catechin, epicatechin and ASEI all decreased gloxal induced hepatocyte cell death and reactive oxygen species (ROS) formation in GSH-depleted hepatocytes. Catechin and epicatechin protected against GO or MGO induced hepatocyte cell death, protein carbonylation and ROS formation. Catechin was more effective than epicatechin. Our results suggest that (a) bioactive almond skin constituents in the non-lipophilic polyphenol extract were the most effective at protecting hepatocytes against hydroperoxide induced hepatocyte oxidative stress and in protecting against dicarbonyl induced cytotoxicity; (b) catechins, the major polyphenol in the extract, were also effective at preventing GO or MGO cytotoxicity likely by trapping GO and MGO and/or rescuing hepatocytes from protein carbonylation.     

Metabolic mechanisms of methanol/formaldehyde in isolated rat hepatocytes: carbonyl-metabolizing enzymes versus oxidative stress

Methanol (CH(3)OH), a common industrial solvent, is metabolized to toxic compounds by several enzymatic as well as free radical pathways. Identifying which process best enhances or prevents CH(3)OH-induced cytotoxicity could provide insight into the molecular basis for acute CH(3)OH-induced hepatoxicity. Metabolic pathways studied include those found in 1) an isolated hepatocyte system and 2) cell-free systems. Accelerated Cytotoxicity Mechanism Screening (ACMS) techniques demonstrated that CH(3)OH had little toxicity towards rat hepatocytes in 95% O(2), even at 2M concentration, whereas 50 mM was the estimated LC(50) (2h) in 1% O(2), estimated to be the physiological concentration in the centrilobular region of the liver and also the target region for ethanol toxicity. Cytotoxicity was attributed to increased NADH levels caused by CH(3)OH metabolism, catalyzed by ADH1, resulting in reductive stress, which reduced and released ferrous iron from Ferritin causing oxygen activation. A similar cytotoxic mechanism at 1% O(2) was previous found for ethanol. With 95% O(2), the addition of Fe(II)/H(2)O(2), at non-toxic concentrations were the most effective agents for increasing hepatocyte toxicity induced by 1M CH(3)OH, with a 3-fold increase in cytotoxicity and ROS formation. Iron chelators, desferoxamine, and NADH oxidizers and ATP generators, e.g. fructose, also protected hepatocytes and decreased ROS formation and cytotoxicity. Hepatocyte protein carbonylation induced by formaldehyde (HCHO) formation was also increased about 4-fold, when CH(3)OH was oxidized by the Fenton-like system, Fe(II)/H(2)O(2), and correlated with increased cytotoxicity. In a cell-free bovine serum albumin system, Fe(II)/H(2)O(2) also increased CH(3)OH oxidation as well as HCHO protein carbonylation. Nontoxic ferrous iron and a H(2)O(2) generating system increased HCHO-induced cytotoxicity and hepatocyte protein carbonylation. In addition, HCHO cytotoxicity was markedly increased by ADH1 and ALDH2 inhibitors or GSH-depleted hepatocytes. Increased HCHO concentration levels correlated with increased HCHO-induced protein carbonylation in hepatocytes. These results suggest that CH(3)OH at 1% O(2) involves activation of the Fenton system to form HCHO. However, at higher O(2) levels, radicals generated through Fe(II)/H(2)O(2) can oxidize CH(3)OH/HCHO to form pro-oxidant radicals and lead to increased oxidative stress through protein carbonylation and ROS formation which ultimately causes cell death.     

Extensive protein carbonylation precedes acrolein-mediated cell death in mouse hepatocytes

Allyl alcohol hepatotoxicity is mediated by an alcohol dehydrogenase-derived biotranformation product, acrolein. This highly reactive alpha,beta-unsaturated aldehyde readily alkylates model proteins in vitro, forming, among other products, Michael addition adducts that possess a free carbonyl group. Whether such damage accompanies acrolein-mediated toxicity in cells is unknown. In this work we established that allyl alcohol toxicity in mouse hepatocytes involves extensive carbonylation of a wide range of proteins, and that the severity of such damage to a subset of 18-50 kDa proteins closely correlated with the degree of cell death. In addition to abolishing cytotoxicity and glutathione depletion, the alcohol dehydrogenase inhibitor 4-methyl pyrazole strongly attenuated protein carbonylation. Conversely, cyanamide, an aldehyde dehydrogenase inhibitor, enhanced cytotoxicity and protein carbonylation. Since protein carbonylation clearly preceded the loss of membrane integrity, it may be associated with the toxic process leading to cell death.     

Hepatocyte or serum albumin protein carbonylation by oxidized fructose metabolites: Glyceraldehyde or glycolaldehyde as endogenous toxins?

Excessive sugar intake in animal models may cause tissue damage associated with oxidative and carbonyl stress cytotoxicity as well as inflammation. Fructose became a 100-fold more cytotoxic if hepatocytes were exposed to a non-toxic infusion of H₂O₂ so as to simulate H₂O₂ released by Kupffer cells or infiltrating immune cells. In order to determine the molecular mechanisms involved, protein carbonylation of fructose and its metabolites were determined using the 2,4-dinitrophenylhydrazine method. In a cell-free system, fructose was found to carbonylate bovine serum albumin (BSA) only if low concentrations of FeII/H₂O₂ were added. Protein carbonylation by the fructose metabolites glyceraldehyde or glycolaldehyde was also markedly increased by FeII/H₂O₂. The protein carbonylation may be attributed to glyoxal formation by hydroxyl radicals as the glyoxal trapping agent aminoguanidine or hydroxyl radical scavengers prevented protein carbonylation. Glyoxal was also much more effective than other carbonyls at causing protein carbonylation. When BSA was replaced by isolated rat hepatocytes, fructose metabolite glyceraldehyde in the presence of non-toxic 2 μM FeII:8-hydroxyquinoline (HQ) and a H₂O₂ generating system (glucose/glucose oxidase) markedly increased cytotoxicity, protein carbonylation and reactive oxygen species (ROS)/H₂O₂ formation. Furthermore this was prevented by hydroxyl radical scavengers or aminoguanidine, a glyoxal scavenger. CuII: 8-hydroxyquinoline increased H₂O₂ induced hepatocyte protein carbonylation less but was prevented by aminoguanidine. However, cytotoxicity and protein carbonylation induced by glyceraldehyde/CuII:HQ/H₂O₂ were not affected by hydroxyl radical scavengers. Although fatty liver induced by an excessive sugar diet in animal models has been proposed as the first hit for non-alcoholic steatohepatitis (NASH) we propose that oxidative stress induced by the oxidation of fructose or fructose metabolites catalysed by Fenton FeII/H₂O₂ could be a ‘second hit’. A perpetual cycle of oxidative stress in hepatocytes could lead to cytotoxicity and contribute to NASH development.     

Cytochrome P450 2E1 metabolically activates propargyl alcohol: propiolaldehyde-induced hepatocyte cytotoxicity

Pargyline, an antihypertensive agent and monoamine oxidase inhibitor, induces hepatic GSH depletion and hepatotoxicity in vivo in rats [E.G. De Master, H.W. Sumner, E. Kaplan, F. N. Shirota, H.T. Nagasawa, Toxicol. Appl. Pharmacol. 65 (1982) 390-401]. Propargyl alcohol (2-propyn-1-ol), because of its structural similarity to allyl alcohol, was thought to be activated by alcohol dehydrogenase. However, it is a poor substrate compared to allyl alcohol and it was therefore proposed that propargyl alcohol-induced liver injury involved metabolic activation by catalase/H(2)O(2) [E.G. De Master, T. Dahlseid, B. Redfern, Chem. Res. Toxicol. 7 (1994) 414-419]. In the following we showed that; (1) propargyl alcohol-induced cytotoxicity was markedly enhanced in CYP 2E1-induced hepatocytes and prevented by various CYP 2E1 inhibitors but was only slightly affected when alcohol dehydrogenase was inhibited with methylpyrazole/DMSO or when catalase was inactivated with azide or aminotriazole, (2) hepatocyte GSH depletion preceded cytotoxicity and was inhibited by cytochrome P450 inhibitors but not by catalase/alcohol dehydrogenase inhibitors. GSH conjugate formation during propargyl alcohol metabolism by microsomal mixed function oxidase in the presence of GSH was also prevented by anti-rat CYP 2E1 or CYP 2E1 inhibitors, (3) cytotoxicity was prevented when lipid peroxidation was inhibited with antioxidants, desferoxamine (ferric chelator) or dithiothreitol. Propargyl alcohol-induced cytotoxicity and reactive oxygen species formation were markedly increased in GSH-depleted hepatocytes. All of this evidence suggests that propargyl alcohol-induced cytotoxicity involves metabolic activation by CYP 2E1 to form propiolaldehyde that causes hepatocyte lysis as a result of GSH depletion and lipid peroxidation.     

Cytochrome P450 2E1 metabolically activates propargyl alcohol: propiolaldehyde-induced hepatocyte cytotoxicity

Pargyline, an antihypertensive agent and monoamine oxidase inhibitor, induces hepatic GSH depletion and hepatotoxicity in vivo in rats [E.G. De Master, H.W. Sumner, E. Kaplan, F. N. Shirota, H.T. Nagasawa, Toxicol. Appl. Pharmacol. 65 (1982) 390–401]. Propargyl alcohol (2-propyn-1-ol), because of its structural similarity to allyl alcohol, was thought to be activated by alcohol dehydrogenase. However, it is a poor substrate compared to allyl alcohol and it was therefore proposed that propargyl alcohol-induced liver injury involved metabolic activation by catalase/H₂O₂ [E.G. De Master, T. Dahlseid, B. Redfern, Chem. Res. Toxicol. 7 (1994) 414–419]. In the following we showed that; (1) propargyl alcohol-induced cytotoxicity was markedly enhanced in CYP 2E1-induced hepatocytes and prevented by various CYP 2E1 inhibitors but was only slightly affected when alcohol dehydrogenase was inhibited with methylpyrazole/DMSO or when catalase was inactivated with azide or aminotriazole, (2) hepatocyte GSH depletion preceded cytotoxicity and was inhibited by cytochrome P450 inhibitors but not by catalase/alcohol dehydrogenase inhibitors. GSH conjugate formation during propargyl alcohol metabolism by microsomal mixed function oxidase in the presence of GSH was also prevented by anti-rat CYP 2E1 or CYP 2E1 inhibitors, (3) cytotoxicity was prevented when lipid peroxidation was inhibited with antioxidants, desferoxamine (ferric chelator) or dithiothreitol. Propargyl alcohol-induced cytotoxicity and reactive oxygen species formation were markedly increased in GSH-depleted hepatocytes. All of this evidence suggests that propargyl alcohol-induced cytotoxicity involves metabolic activation by CYP 2E1 to form propiolaldehyde that causes hepatocyte lysis as a result of GSH depletion and lipid peroxidation.     

Aldehyde dehydrogenase heterogeneity in rat hepatic cells

In normal rat liver, aldehyde dehydrogenase (Aldehyde:NAD+ oxidoreductase, EC 1.2.1.3; ALDH) is found primarily in mitochondrial and microsomal fractions. During hepatocarcinogenesis, an additional tumor-associated aldehyde dehydrogenase (T-ALDH) is detectable in the cytosol of preneoplastic and neoplastic cells. We report here differences in the ALDH distribution pattern in different rat hepatoma cell lines compared to normal rat hepatocytes. Of the four basal ALDH enzymes, one mitochondrial ALDH and one microsomal ALDH account for 96% of total ALDH molecules detectable with our probes in normal hepatocytes. The other two mitochondrial and microsomal ALDH enzymes are only detectable in the appropriate subcellular fraction from large populations of cells. The tumor-associated ALDH is not detectable in normal hepatocytes. In addition to varying amounts of T-ALDH in the six different rat hepatoma cell lines examined, differences in the amounts of mitochondrial and microsomal ALDHs also occur in both high and low T-ALDH activity hepatoma cell lines. Each of five ALDH enzymes examined has a characteristic half-life varying from 45 min to 95 h.     

Enhancement of aldehyde dehydrogenase activity in human and rat hepatocyte cultures by 3-methylcholanthrene

Aldehyde dehydrogenase was measured in primary cultures of hepatocytes obtained with a two-step collagenase perfusion either from human hepatic tissue or from livers of Fisher rats. Basal enzyme activity declines gradually as a function of time in culture, but remains at all times higher when measured with propionaldehyde and NAD (P/NAD) than with benzaldehyde and NADP (B/NADP). Treatment of the cultures with 2 M of 3-methylcholanthrene for four days significantly increased the B-NADP activity of human and rat hepatocytes (tenfold and eightfold respectively). In human hepatocytes 3-methylcholanthrene increases also the P/NAD activity, but to a lesser extent (twofold), compared to the B/NADP activity. Due to the significant enhancement of B/NADP activity in cultures of human and rat hepatocytes after application of 3-methylcholanthrene, the initial difference in the basal activity levels between the P/NAD and B/NADP forms diminishes or, in the case of human hepatocytes, is even inverted. These results show for the first time that aldehyde dehydrogenase activity is increased in cultured human hepatocytes. This biochemical property is preserved in human and rat hepatocyte cultures, despite the rather quick loss of the basal aldehyde dehydrogenase activity.Abbreviations ALDH aldehyde dehydrogenase - B benzaldehyde - p-p-DDT 1,1,1,-trichlo-2,2,bis(p-chlorophenyl)ethane - DMSO dimethylsulfoxide - 3-MC 3-methylcholanthrene - MEM Minimal Essential Medium - P proprionaldehyde - TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin     

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.     

The antihypertensive hydralazine is an efficient scavenger of acrolein

Recent work indicates the highly toxic alpha,beta-unsaturated aldehyde acrolein is formed during the peroxidation of polyunsaturated lipids, raising the possibility that it functions as a 'toxicological second messenger' during oxidative cell injury. Acrolein reacts rapidly with proteins, forming adducts that retain carbonyl groups. Damage by this route may thus contribute to the burden of carbonylated proteins in tissues. This work evaluated several amine compounds with known aldehyde-scavenging properties for their ability to attenuate protein carbonylation by acrolein. The compounds tested were: (i) the glycoxidation inhibitors, aminoguanidine and carnosine; (ii) the antihypertensive, hydralazine; and (iii) the classic carbonyl reagent, methoxyamine. Each compound attenuated carbonylation of a model protein, bovine serum albumin, during reactions with acrolein at neutral pH and 37 degrees C. However, the most efficient agent was hydralazine, which strongly suppressed carbonylation under these conditions. Study of the rate of reaction between acrolein and the various amines in a protein-free buffered system buttressed these findings, since hydralazine reacted with acrolein at rates 2-3 times faster than its reaction with the other scavengers. Hydralazine also protected isolated mouse hepatocytes against cell killing by allyl alcohol, which undergoes in situ alcohol dehydrogenase-catalysed conversion to acrolein.     

Hydrogen peroxide-induced carbonylation of key metabolic enzymes in Saccharomyces cerevisiae: the involvement of the oxidative stress response regulators Yap1 and Skn7

H(2)O(2) induces a specific protein oxidation in yeast cells, and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (Tdh) is a major target. Using a 2D-gel system to study protein carbonylation, it is shown in this work that both Tdh2p and Tdh3p isozymes were oxidized during exposure to H(2)O(2). In addition, we identified two other proteins carbonylated and inactivated: Cu,Zn-superoxide dismutase and phosphoglycerate mutase. The oxidative inactivation of Cu,Zn-superoxide dismutase decreases the antioxidant capacity of yeast cells and probably contributes to H(2)O(2)-induced cell death. Cyclophilin 1 was also carbonylated, but CPH1 gene disruption did not affect peroxide stress sensitivity. The correlation between H(2)O(2) sensitivity and the accumulation of oxidized proteins was evaluated by assaying protein carbonyls in mutants deficient in the stress response regulators Yap1p and Skn7p. The results show that the high sensitivity of yap1delta and skn7delta mutants to H(2)O(2) was correlated with an increased induction of protein carbonylation. In wild-type cells, the acquisition of stress resistance by pre-exposure to a sublethal H(2)O(2) stress was associated with a lower accumulation of oxidized proteins. However, pre-exposure of yap1delta and skn7delta cells to 0.4 mM H(2)O(2) decreased protein carbonylation induced by 1.5 mM H(2)O(2), indicating that the adaptive mechanism involved in the protection of proteins from carbonylation is Yap1p- and Skn7p-independent.     

Enhanced 5-HT<Subscript>2A</Subscript> Receptors in Brain Stem and ALDH Activity in Brain Stem and Liver: 5-HT<Subscript>2A</Subscript> Regulation on ALDH in Primary Hepatocytes Cultures In Vitro

Brain serotonin (5-HT) modulates the neural effects of ethanol. In the present study, we investigated the changes in 5-HT level, 5-HT_2A receptor binding and aldehyde dehydrogenase (ALDH) activity in brain stem and liver of ethanol treated rats and 5-HT_2A regulation on ALDH in hepatocyte cultures in vitro. The 5-HT content in the brain stem and liver significantly decreased with an increased 5-HIAA/5-HT ratio in the ethanol treated rats compared to control. Scatchard analysis of [³H] (±)2,3-dimethoxyphenyl-1-[2-(-4-piperidine)-methanol] [³H] MDL 100907 against ketanserin in brain stem of ethanol treated rats showed a significant increase in B _max without any change in K _d compared to control. The competition curve for [³H] MDL 100907 against ketanserin fitted one-site model in both control and ethanol treated rats with unity as Hill slope value. A significant increase in V _max of ALDH activity in liver and a significant decrease in K _m in liver and brain stem of ethanol treated rats compared to control was observed. In 24 h culture studies, an increase in enzyme activity was observed in cells in medium with 10% ethanol. The elevated ALDH activity in ethanol treated cells was reversed to control level in presence of 10⁻⁵ and 10⁻⁷ M 5-HT. Ketanserin, an antagonist of 5-HT_2A, reversed the effect of 5HT on 10% ethanol induced ALDH activity in hepatocytes. Our results showed that there was a decreased 5-HT content with an enhanced 5-HT_2A receptor and aldehyde dehydrogenase activity in the brain stem of alcohol treated rats and in vitro hepatocyte cultures. The enhanced ALDH activity in ethanol supplemented hepatocytes was reversed to control level in presence of 10⁻⁵ and 10⁻⁷ M 5-HT.     

The molecular mechanisms of diallyl disulfide and diallyl sulfide induced hepatocyte cytotoxicity

Diallyl disulfide (DADS) and diallyl sulfide (DAS) are the major metabolites found in garlic oil and have been reported to lower cholesterol and prevent cancer. The molecular cytotoxic mechanisms of DADS and DAS have not been determined.The cytotoxic effectiveness of hydrogen versus allyl sulfides towards hepatocytes was found to be as follows: NaHS > DADS > DAS. Hepatocyte mitochondrial membrane potential was decreased and reactive oxygen species (ROS) and TBARS formation was increased by all three allyl sulfides. (1) DADS induced cytotoxicity was prevented by the H₂S scavenger hydroxocobalamin, which also prevented cytochrome oxidase dependent mitochondrial respiration suggesting that H₂S inhibition of cytochrome oxidase contributed to DADS hepatocyte cytotoxicity. (2) DAS cytotoxicity on the other hand was prevented by hydralazine, an acrolein trap. Hydralazine also prevented DAS induced GSH depletion, decreased mitochondrial membrane potential and increased ROS and TBARS formation. Chloral hydrate, the aldehyde dehydrogenase 2 inhibitor, however had the opposite effects, which could suggest that acrolein contributed to DAS hepatocyte cytotoxicity.     

3,4-Dihydroxyphenylacetic acid is a potential aldehyde dehydrogenase inducer in murine hepatoma Hepa1c1c7 cells

3,4-Dihydroxyphenylacetic acid (DOPAC) is one of the major colonic microflora-produced catabolites of quercetin glycosides, such as quercetin 4′-glucoside derived from onion. Here, we investigated whether DOPAC modulates the aldehyde dehydrogenase (ALDH) activity and protects the cells from the acetaldehyde-induced cytotoxicity in vitro. DOPAC was shown to enhance not only the total ALDH activity, but also the gene expression of ALDH1A1, ALDH2 and ALDH3A1 in a concentration-dependent manner. DOPAC simultaneously stimulated the nuclear translocation of NFE2-related factor 2 and aryl hydrocarbon receptor. The pretreatment of DOPAC completely protected the cells from the acetaldehyde-induced cytotoxicity. The present study suggested that DOPAC acts as a potential ALDH inducer to prevent the alcohol-induced abnormal reaction.     

Proteomic identification of mitochondrial carbonylated proteins in two maturation stages of pepper fruits

Pepper fruits in green and red maturation stages were selected to study the protein pattern modified by oxidation measuring carbonylated proteins in isolated mitochondria, together with the accumulation of superoxide radical and hydrogen peroxide in the fruits. MALDI‐TOF/TOF analysis identified as carbonylated proteins in both green and red fruits, formate dehydrogenase, NAD‐dependent isocitrate dehydrogenase, porin, and defensin, pointing to a common regulation by carbonylation of these proteins independently of the maturation stage. However, other proteins such as glycine dehydrogenase P subunit and phosphate transporter were identified as targets of carbonylation only in green fruits, whereas aconitase, ATPase β subunit, prohibitin, orfB protein, and cytochrome C oxidase, were identified only in red fruits. In general, the results suggest that carbonylation of mitochondrial proteins is a PTM that drives the complex ripening process, probably establishing the accumulation and functionality of some mitochondrial proteins in the nonclimacteric pepper fruit.     

Antioxidant and pro-oxidant actions of resveratrol on human serum albumin in the presence of toxic diabetes metabolites: Glyoxal and methyl-glyoxal

Methylglyoxal (MGO) and glyoxal (GO) are attracting considerable attention because of their role in the onset of diabetes symptoms. Therefore, to comprehend the molecular fundamentals of their pathological actions is of the utmost importance. In this study, the molecular interactions between resveratrol (RES) and human serum albumin (HSA) and the ability of the stilbene to counteract the oxidative damage caused by pathological concentrations of MGO and GO to the human plasma protein, was assessed. The oxidation of Cys34 in HSA as well as the formation of specific protein semialdehydes AAS (α-aminoadipic), GGS (γ-glutamic) and the accumulation of Advanced Glycation End-products (AGEs) was investigated. Resveratrol was found to neutralize both α-dicarbonyls by forming adducts detected by HESI-Orbitrap-MS. This antioxidant action was manifested in a significant reduction of AGEs. However, RES-α-dicarbonyl conjugates oxidized Cys34 and lysine, arginine and/or proline by a nucleophilic attack on SH and ε-NH groups in HSA. The formation of specific semialdehydes in HSA after incubation with GO and MGO at pathological concentrations was reported for the first time in this study, and may be used as early and specific biomarkers of the oxidative stress undergone by diabetic patients. The pro-oxidative role of the RES-α-dicarbonyl conjugates should be further investigated to clarify whether this action leads to positive or harmful clinical consequences. The biological relevance of human protein carbonylation as a redox signaling mechanism and/or as a reflection of oxidative damage and disease should also be studied in future works.     

Chromosomal assignment of the genes for human aldehyde dehydrogenase-1 and aldehyde dehydrogenase-2.

Chromosomal assignment of the genes for two major human aldehyde dehydrogenase isozymes, that is, cytosolic aldehyde dehydrogenase-1 (ALDH1) and mitochondrial aldehyde dehydrogenase-2 (ALDH2) were determined. Genomic DNA, isolated from a panel of mouse-human and Chinese hamster-human hybrid cell lines, was digested by restriction endonucleases and subjected to Southern blot hybridization using cDNA probes for ALDH1 and for ALDH2. Based on the distribution pattern of ALDH1 and ALDH2 in cell hybrids, ALDH1 was assigned to the long arm of human chromosome 9 and ALDH2 to chromosome 12.     

Pattern of occurrence and occupancy of carbonylation sites in proteins

Proteins are targets for modification by reactive oxygen species, and carbonylation is an important irreversible modification that increases during oxidative stress. While information on protein carbonylation is accumulating, its pattern is not yet understood. We have made a meta-analysis of the available literature data (456 carbonylation sites on 208 proteins) to appreciate the nature of carbonylation sites in proteins. Of the carbonylated (Arg, Lys, Pro, and Thr - RKPT) amino acids, Lys is the most abundant, whereas Pro is the most susceptible and Thr is the least susceptible. The incidence of carbonylation is lower in the N-terminal part of the protein primary sequence. Although a significantly higher number of carbonylated sites occur in Arg-, Lys-, Pro- and Thr-rich regions of proteins, the hydropathy environment of carbonylated sites is not significantly different from potential carbonylation sites. Comparison of metal-catalyzed oxidation of two closely related proteins indicates that this type of carbonylation might not be very specific in proteins. Interestingly, carbonylated sites show a very strong tendency to cluster together in the protein primary sequence hinting at some sort of discerning mechanism. While some attributes of protein carbonylation appear to be random, further investigations are warranted to appreciate the deterministic nature of protein carbonylation sites.