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Studies on the efficacy of lopoate and dihydrolipoate in the alteration of Cd2± toxicity in isolated hepatocytes
Abstract
Lipoate (thioctic acid) is presently used in therapy of a variety of diseases such as liver and neurological disorders. However, nothing is known about the efficacy of lipoate and its reduced form dihydrolipoate in acute cadmium (Cd2+) toxicity which involves severe liver disturbances. Therefore, we investigated the effects of these redox compounds on Cd2(+)-induced injuries in isolated rat hepatocytes. The cells were coincubated with 150 microM Cd2+ and either 1.5-6.0 mM lipoate or 17-89 microM dihydrolipoate for up to 90 min and Cd2+ uptake as well as viability criteria were monitored. Both exposure regimens diminished Cd2+ uptake in correspondence to time and concentration. They also ameliorated Cd2(+)-induced cell deterioration as reflected by the decrease in Cd2(+)-induced membrane damage (leakage of aspartate aminotransferase), by the lessening of the Cd2(+)-stimulated lipid peroxidation (TBA-reactants) and by the increase in Cd2(+)-depleted cellular glutathione (GSH + 2 GSSG). Half-maximal protection was achieved at molar ratios of 9.9 to 19 (lipoate vs. Cd2+) and 0.25 to 0.74 (dihydrolipoate vs. Cd2+), indicating a 19.5 to 50.6 lower protective efficacy of lipoate as compared to dihydrolipoate. Lipoate induced an increase in extracellular acid-soluble thiols different from glutathione. It is suggested that dihydrolipoate primarily protects cells by extracellular chelation of Cd2+, whereas intracellular reduction of lipoate to the dihydro-compound followed by complexation of both intra- and extracellular Cd2+ contributes to the amelioration provided by lipoate.
... It is essential for the proper functioning of several enzymes that play a key role in oxidative metabolism [1][2]. In particular, it is a powerful antioxidant that is soluble in both water and fat; thus, it is easily absorbed after oral administration [3][4][5][6]. ...
Introduction
Peripheral neuropathy is one of the possible complications of diabetes. Alpha-lipoic acid (a-lipoic acid or ALA) is a powerful antioxidant cofactor synthesized in mitochondria that could help stimulate nerves and regenerate nerve fibers, thus preventing disease progression. Moreover, the possible feeling of oppression from the lifestyle changes needed to avoid the complications of diabetes may contribute to the development of depressive symptoms. ALA increases insulin sensitivity, which could increase serotonin synthesis and thus reduce the manifestations of depressive disorder.
Aim
The aim of this study is to investigate the therapeutic effect after oral administration of a-lipoic acid in patients with type II diabetes mellitus, regarding the possibility of developing peripheral neuropathy and the possibility of developing depressive disorder due to the existence of diabetes type II.
Methods
The study sample consisted of 148 Greek patients, type II diabetics, 68 men and 80 women, aged 50-75 years. All of them were non-smokers and did not consume alcohol. Their treatment was a combination of gliclazide, sodium-glucose-linked transporter 2 (SGLT-2) inhibitors, metformin, and glucagon-like peptide 1 (GLP-1) analogs. None of them were under insulin administration. Any other treatment received chronically from the patients for other comorbidities was not altered or paused. All patients were in regular monitoring of renal, hepatic, and ocular function, which was normal. Patients were monitored with a balanced diet, based on equivalents, in order to maintain an almost constant body mass index (BMI). All were given one tablet of 600 mg a-lipoic acid, two hours before a meal, for eight months, and the incidence of developing peripheral neuropathy and depressive disorder was assessed, using the Subjective Peripheral Neuropathy Screen Questionnaire (SPNSQ) and Beck Depression Inventory (BDI) questionnaire.
Results
ALA administration after both four and eight months resulted in statistically significant results and, specifically, the peripheral neuropathy development mean score was reduced by 4.79 at four months and 6.22 after eight months. Concerning the incidence of depressive disorder, an average decrease of 4.43 in the related depression score was observed at the four-month milestone and 7.56 at eight months, both statistically significant.
Conclusion
A-lipoic acid is a powerful antioxidant and, when used with conventional treatment, has shown to significantly decrease the incidence of depression and peripheral neuropathy in patients with type 2 diabetes mellitus.
... Thus, these reactions can be responsible for the formation of ROS in the body. Therefore, the chelation of Fe and other metals can be helpful to the antioxidant impact [38]. ...
Background:
We have investigated the effects of α-lipoic acid (LA), a powerful antioxidant, on the fatty acid (FA) profiles, aluminum accumulation, antioxidant activity and some minerals such as zinc, copper and iron against aluminum chloride (AlCl3)-induced oxidative stress in rat liver.
Methods:
Twenty-eight male Wistar rats were divided into four groups as control, LA, AlCl3 and LA+AlCl3. For 30 days, LA was intraperitoneally administrated (50 mg/kg) and AlCl3 was given via orogastric gavage (1600 ppm) every other day.
Results:
AlCl3-treated animals exhibited higher hepatic malondialdehyde concentration and lower glutathione peroxidase and catalase activity, whereas these alterations were restored by the LA supplementation. Total saturated FA of the AlCl3-treated group was higher than the LA supplementation groups. Moreover, total unsaturated FA level of the LA+AlCl3 group was higher than the AlCl3-treated group. Hepatic zinc level of the AlCl3-treated group was lower than the control group, whereas it was higher in the LA and the LA+AlCl3 groups. Hepatic copper levels did not significantly change in the experimental groups. Iron level was lower in the LA and LA+AlCl3 groups compared with the AlCl3-treated group. Moreover, the liver Al concentration was found to be lower in the LA and LA+AlCl3 groups compared to the AlCl3 group.
Conclusions:
These results indicate that AlCl3 treatment can induce oxidative stress in the liver. LA supplementation has a beneficial effect on the AlCl3-induced alterations such as high lipid peroxidation, Al accumulation, FA profile ratios and mineral concentrations.
... These antioxidant properties led to a widespread use of racemic LA or R(þ)-LA as dietary supplements (Berkson 1998;Sosin and Ley-Jacobs 1998;Packer and Colman 2000). Yet, results of numerous studies and clinical trials on the preferred mode of administration, effective dosage, potency, efficacy, and the pharmacokinetics of LA often remain inconsistent (Muller and Menzel 1990;Jacob et al. 1996;Streeper et al. 1997;Hagen et al. 1999;Jacob et al. 1999;Konrad et al. 1999;Ruhnau et al. 1999;Evans and Goldfine 2000;Suh et al. 2001;Moini et al. 2002;Frolich et al. 2004;Hahm et al. 2004;Ziegler 2004;Ziegler et al. 2004). Generally, the pharmacokinetic analysis of LA suggests that intravenous administration of LA reaches and sustains effective concentrations of the compounds in the plasma of treated individuals better than oral administration of similar or even larger doses (Evans and Goldfine 2000). ...
... 26 In another study, alpha lipoic acid was demonstrated to be able to protect rat hepatocytes from cadmium toxicity by preventing decreases in total glutathione and increases in lipid peroxidation. 27 These protective effects have also been seen in rats that had depleted levels of glutathione prior to cadmium exposure. 28 A study with rats examined the effects of alpha lipoic acid on arsenic toxicity. ...
... Concentration of As 3+ was chosen based on prior studies of our group where 50 M As 3+ resulted in a minimal reduction in cell viability on HepG2 cells and still was able to induce a strong expression of stress genes and related proteins. Concentration of ALA was the same as used by Muller and Menzel (1990) which was able to protect rat hepatocytes from cadmium toxicity and also showed no signs of toxicity on HepG2 cells in previous experiments by our group. After exposure for 8 h, cells were washed twice with PBS and collected for analysis of MTT-metabolic activity, GSH levels, analysis of gene expression of several stress and antioxidant-related genes and Nrf2 induction. ...
... Lipoic acid does not act like the tocopherols, in that it does not show chain-breaking properties in lipids in vitro (Biewenga et al., 1997). Lipoic acid is effective at chelating transition metals, such as cadmium, although DHLA may have been involved (Muller and Menzel, 1990). The beta pentanoic side-chain can undergo oxidation in man to bisnorlipoic acid (Biewenga et al., 1997) and this agent also has reductive properties. ...
Lipoic acid and its reduced derivative, dihydrolipoic acid (DHLA) are highly promising antioxidant agents, which are potent attenuators of reactive species-mediated damage in vitro and in animal studies. Lipoic acid is a universal antioxidant, effective in lipophilic and aqueous environments. In contrast to an equivalent endogenous agent, such as oxidised glutathione (GSSG), lipoic acid acts as an antioxidant in its oxidised form. Lipoic acid has been evaluated in diabetic polyneuropathy, a condition which is thought to result in part from oxidant damage caused by long-term hyperglycaemia. Diabetic patients are prone to incur enhanced cellular free radical formation and reduced antioxidant defence. Treatment with lipoic acid has improved nerve conduction velocity during studies in diabetic animals. Trials in diabetic patients have often observed some relief of neuropathic symptoms during treatment with lipoic acid, but consistent objective benefits have been difficult to establish. Lipoic acid is now used in Germany for the treatment of diabetic neuropathy and definitive evidence of efficacy should arise from postmarketing surveillance studies. It is possible that lipoic acid may be more effective as a long-term dietary supplement aimed at the prophylactic protection of diabetics from complications.
... In addition, ␣-Lipoic acid protected cells from single-strand DNA breaks and scavenged singlet oxygen produced following endoperoxide thermolysis [39]. ␣-Lipoic acid also decreases Cd 2ϩ toxicity and forms stable complexes with Mn 2ϩ , Cu 2ϩ , and Zn 2ϩ [40,41]. However, ␣-Lipoic acid does not seem to protect liver microsomes from Fe-induced lipid peroxidation [42]. ...
Lead (Pb) is known to negatively affect glutathione (GSH) metabolism in the lens. The present study examined the effects of Captopril, Taurine, and α-Lipoic acid on the Pb-induced GSH depletion and lipid peroxide increase in the lenticular system. Captopril administration returned the GSH, cysteine (CYS), and malondialdehyde (MDA) levels to near normal. Following Taurine administration the GSH, CYS and MDA levels were intermediate between the control group and the Pb group levels. α-Lipoic acid administration, however, only increased the CYS levels. No significant changes in oxidized glutathione (GSSG) levels were observed in any treatment group.
... A lipophilic complex of LA with Cu 2+ has also been suggested to explain protection in Cu 2+ -induced lipid peroxidation [38]. DHLA can act in the same way by chelating Cd 2+ [36] and Cu 2+ [31]. The chelating capacity of DHLA is more effective than that of LA, but it has been found to have also pro-oxidant properties [31]. ...
Lipoic acid (1,2-dithiolane-pentanoic acid) is a dithiol which is effective in affording protection against oxidative stress by virtue of its two sulphydryl moieties. It is present in all kinds of eukaryotic and prokaryotic cells. As lipoamide, it functions as a cofactor in the multienzyme complexes that catalyse the oxidative decarboxylation of α-keto acids such as pyruvate, α-ketoglutarate, and branched-chain α-keto acids. The complete enzyme pathway responsible for the de novo synthesis of lipoic acid has not yet been elucidated. Octanoic acid appears to be the precursor for the eight-carbon fatty acid chain, and cysteine the source of sulfur. Lipoic acid is unique, among antioxidants, because it retains powerful antioxidant properties in both its reduced (dihydrolipoic acid) and oxidised (lipoic acid) forms. Both lipoic and dihydrolipoic acids have metal-chelating ability and quench activated oxygen species either in the cytosol or in the hydrophobic domains. Dihydrolipoic acid has more antioxidant properties than lipoic acid, and it plays an important role in the recycling of other oxidised radical scavengers such as glutathione, ascorbate and tocopherol. However, dihydrolipoic acid can also exert pro-oxidant properties both by its iron-reducing ability and by its ability to generate sulfur-containing radicals that can damage proteins. There are few quantitative data on lipoic acid contents in vegetables. It has been found in asparagus, wheat and potatoes, and recently, the presence of both lipoic and dihydrolipoic acids in roots, leaves and in the stroma of wheat has been demonstrated.
... Chemically synthesized LA, in contrast, is a racemate (a mixture of equal amount of R-and S-LA), and is believed to exert additional beneficial bioactivities when administered exogenously. These include anti-oxidative activities such as chelating metals (Muller et al., 1990), scavenging reactive oxygen species (ROS) (Matsugo et al, 1995), recycling / inducing endogenous anti-oxidants (Konishi et al., 1996), anti-inflammation (Fuchs et al., 1994), activation of cell signaling (Packer et al., 2011), and enhancement of glucose uptake (Konrad et al., 2001). It was also reported that LA had a pro-oxidant activity which leads to cellular adaptation against oxidative stress (Cakatay, 2006). ...
Growing evidence suggests that α-lipoic acid (LA) has neuroprotective effects in various pathological conditions including brain ischemia and neurodegeneration. While anti-oxidative activity has been thought to play a central role in LA-mediated neuroprotection, the precise mechanism and the effect of LA enantiomers (R- and S-LA) are not fully clarified. We, therefore, estimated the neuroprotective effects of LA against different cellular stresses including oxidative stress, endoplasmic reticulum (ER) stress and proteolytic stress using human neuroblastoma SH-SY5Y cells. All types of LAs (racemate, R-LA and S-LA) most effectively prevented cell death induced by buthionine sulfoximine (BSO) which depletes intracellular glutathione. Although direct effects of LA on glutathione depletion or generation of the reactive oxygen species (ROS) were relatively small upon BSO treatment, LA enhanced expressions of anti-oxidative genes such as heme oxygenase-1 (HO-1) and phase II detoxification enzymes such as NAD(P)H:Quinone Oxidoreductase 1 (NQO1). An inhibitor of NQO1, but not that of HO-1, suppressed LA-mediated protection against BSO. Further experiments revealed that all types of LAs activated cell survival-associated kinase Akt, and an inhibitor of PI3K, LY294002, suppressed both LA-induced upregulation of NQO1 and cell protection against BSO. Our results suggest an important role of PI3K/Akt-mediated upregulation of genes including phase II enzymes such as NQO1 in LA-mediated neuroprotection.
... Concentration of As 3+ was chosen based on prior studies of our group where 50 M As 3+ resulted in a minimal reduction in cell viability on HepG2 cells and still was able to induce a strong expression of stress genes and related proteins. Concentration of ALA was the same as used by Muller and Menzel (1990) which was able to protect rat hepatocytes from cadmium toxicity and also showed no signs of toxicity on HepG2 cells in previous experiments by our group. After exposure for 8 h, cells were washed twice with PBS and collected for analysis of MTT-metabolic activity, GSH levels, analysis of gene expression of several stress and antioxidant-related genes and Nrf2 induction. ...
Oxidative stress is a known mechanism induced, among other things, by arsenic toxicity. As a response, the cell triggers the synthesis of antioxidant and stress response elements like glutathione and heme oxygenase. Alpha-lipoic acid (ALA) is a well-known antioxidant that confers protection to oxidative stress conditions. We analyzed the effect of ALA pretreatment on Nrf2-responsive gene expression of HepG2 cells exposed to As(3+). Cells were treated with 5mM ALA and 8h later exposed to 50μM As(3+) for 24h, analyzing MTT-activity, glutathione content, Nrf2 induction and antioxidant gene expression. As(3+) increased glutathione (154%), heme oxygenase, glutamate cystein ligase, modifier subunit and metallothionein (35-fold, 10-fold and 9-fold, respectively). ALA prevented the strong expression of heme oxygenase by As(3+) exposure (from 35- to 5-times of control cells), which correlated with the reduction of Nrf2 observed in As(3+) group. ALA pretreatment can down-modulate the response mediated by Nrf2 and provide protection to As(3+) exposed HepG2 cells.
... Lipoic acid and its reduced form dihydrolipoic acid are present in all kinds of microbial and eukaryotic cells and act as antioxidants not only through free-radical quenching, but also indirectly through recycling other cellular antioxidants (8). Its potential as a chelating agent against heavy metal poisoning was also evaluated by some laboratories (9,10). ...
We investigated the muscle tissue of a teleost Cyprinus carpio L. to find out whether N-acetylcysteine (NAC), alpha-lipoic acid (LA), taurine (TAU), and curcumin (CUR) were able to counteract oxidative stress induced by acute exposure to cadmium (Cd). The muscle tissue was dissected 96 h after a single intraperitoneal injection of Cd (5 mg kg(-1)) and of antioxidant substances (50 mg kg(-1)). Using spectrophotometry, we determined the glutathione redox status, lipid peroxidation levels and the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione disulphide reductase (GR). Accumulation of Cd in the muscle was analysed using inductively coupled plasma - optical emission spectrometry (ICP-OES).All substances lowered Cd levels in the following order of efficiency; LA=NAC>TAU=CUR. Cadmium increased SOD activity, but CAT activity declined, regardless of antioxidant treatment. Treatment with CUR induced GPx activity. Treatment with TAU lowered Cd due to higher total glutathione (tGSH). The most effective substances on lipid peroxidation were LA and NAC due to a greater Cd-lowering potential. It seems that the protective role of TAU, LA, and NAC is not necessarily associated with antioxidant enzymes, but rather with their own activity.
... Lipoic acid and its reduced form dihydrolipoic acid are present in all kinds of microbial and eukaryotic cells and act as antioxidants not only through free-radical quenching, but also indirectly through recycling other cellular antioxidants (8). Its potential as a chelating agent against heavy metal poisoning was also evaluated by some laboratories (9,10). ...
We investigated the muscle tissue of a teleost Cyprinus carpio L. to find out whether N-acetylcysteine (NAC), alpha-lipoic acid (LA), taurine (TAU), and curcumin (CUR) were able to counteract oxidative stress induced by acute exposure to cadmium (Cd). The muscle tissue was dissected 96 h after a single intraperitoneal injection of Cd (5 mg kg-1) and of antioxidant substances (50 mg kg-1). Using spectrophotometry, we determined the glutathione redox status, lipid peroxidation levels and the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione disulphide reductase (GR). Accumulation of Cd in the muscle was analysed using inductively coupled plasma - optical emission spectrometry (ICP-OES). All substances lowered Cd levels in the following order of effi ciency; LA=NAC>TAU=CUR. Cadmium increased SOD activity, but CAT activity declined, regardless of antioxidant treatment. Treatment with CUR induced GPx activity. Treatment with TAU lowered Cd due to higher total glutathione (tGSH). The most effective substances on lipid peroxidation were LA and NAC due to a greater Cd-lowering potential. It seems that the protective role of TAU, LA, and NAC is not necessarily associated with antioxidant enzymes, but rather with their own activity.
... LA has been intensely investigated as a therapeutic agent in a number of conditions related to liver disease, including alcohol-induced damage, mushroom poisoning, Cd 2+ -intoxification, CCl 4 poisoning, and hyperdynamic circulation in biliary cirrhosis (Cohen M.R. et al., 1971;Muller and Menzel, 1990;Bludovska et al., 1999;Vancini B., 1959;Marley et al., 1999;Bustamante et al., 1998). Beyond the antioxidative properties of LA, however, few is known about potential cellular and molecular targets of LA in liver disease. ...
107 Ischemia reperfusion injury (IRI) represents a major clinical problem in liver transplantation and resection. Main pathophysiological events during this injury comprise depletion of ATP, Kupffer cell activation with subsequent formation of reactive oxygen species and inflammatory response. Depending on the severity of IRI, cell damage leads to necrotic or apoptotic liver cell death, resulting in organ dys- or even nonfunction. The present work investigated the potential of α-lipoic acid (LA), a well established agent in the therapy of diabetic polyneuropathy, to reduce IRI of the rat liver. In the system of the isolated perfused rat liver, an experimental model of hepatic IRI, LA was utilized in different treatment protocols and concentrations. As a key result of this study, pre- as well as postischemic treatment with LA was shown for the first time to markedly reduce hepatic IRI. Further investigations characterized the underlying mechanisms of LA action, focussing on the preconditioning protocol. Thereby, LA was shown to increase hepatic ATP content during ischemia and reperfusion, suggesting a better energy availability in these organs. Activation of the redox-sensitive transcription factors NF-κB and AP-1 was reduced by LA, suggesting a decreased inflammatory response of the liver. Most importantly, this work describes for the first time that the protein kinase Akt plays a crucial role in IRI. The cytoprotective kinase was activated by LA preconditioning during ischemia and reperfusion. Blocking Akt activation by simultaneous application of wortmannin, a selective PI-3 kinase inhibitor, abrogated the LA preconditioning effect, showing a causal involvement of Akt in LA-mediated protection from IRI. Therefore, Akt activation can be regarded as a new protective mechanism in hepatic IRI. To conclude, LA treatment might represent a new pharmacological approach in attenuating IRI of the liver.
... The importance of any fluctuations in NADH/NAD + following supplementation would be critical to understanding of the mechanism(s) behind lipoic acid. Both lipoic acid and its reduced counterpart, DHLA have been effective against oxidative stress [76]. It has been suggested that lipoic acid is transported across the mitochondrial membrane for reduction and subsequently imported into the mitochondrial compartment as DHLA, at the expense of mitochondrial-reducing equivalents such as NADH and NADPH [38,103]. ...
This thesis investigated whole body glucose disposal and the adaptive changes in skeletal muscle carbohydrate metabolism following 28 d of supplementation with 1000 mg R(+)-lipoic acid in young sedentary males (age, 22.1 ± 0.67 yr, body mass, 78.7 ± 10.3 kg, n=9). In certain individuals, lipoic acid decreased the 180-min area under the glucose concentration and insulin concentration curve during an oral glucose tolerance test (OGTT) (n=4). In the same individuals, lipoic acid supplementation decreased pyruvate dehydrogenase kinase activity (PDK) (0.09 ± 0.024 min"^ vs. 0.137 ± 0.023 min'\ n=4). The fasting levels of the activated form of pyruvate dehydrogenase (PDHa) were decreased following lipoic acid (0.42 ± 0.13 mmol-min'kg'^ vs. 0.82 ± 0.32 mmolrnin'^kg"\ n=4), yet increased to a greater extent during the OGTT (1.21 ± 0.34 mmol-min'kg"' vs. 0.81 ±0.13 mmolmin"'kg'\ n=4) following hpoic acid supplementation. No changes were demonstrated in the remaining subjects (n=5). It was concluded that improved glucose clearance during an OGTT following lipoic acid supplementation is assisted by increased muscle glucose oxidation through increased PDHa activation and decreased PDK activity in certain individuals.
... Both α-lipoic acid and DHLA may chelate or bind metal ions and facilitating their removal from the cell (Ou et al., 1995 ). Exogenous administration of α-lipoic acid has been found to be effective in many pathological condition associated with oxidative stress, diabetic neuropathy (Zeigler et al., 1999), metal toxicity (Muller and Menzel, 1990 ), hypertension (Midaoui and Champlain, 2002), diabetic complication and cataracts (Packer, 1994). Recently it has been found that α-lipoic acid suppressed the free radicals initiated by arsenic in different parts of rat brain regions (Shila et al., 2005a). ...
The patient of chronic arsenic toxicity shows oxidative stress. To overcome the oxidative stress, several antioxidants such as beta-carotene, ascorbic acid, α-tocopherol, zinc and selenium had been suggested in the treatment of chronic arsenic toxicity. In the present study universal antioxidant (both water and lipid soluble antioxidant) α-lipoic acid was used to examine the effectiveness of reducing the amount of arsenic from arsenic-loaded isolated liver tissues of rat. Isolated liver tissues of Long Evans Norwegian rats were cut into small pieces and incubated first in presence or absence of arsenic and then with different concentrations of α-lipoic acid during the second incubation. α-Lipoic acid decreases the amount of arsenic and malondialdehyde (MDA) in liver tissues as well as increases the reduced glutathione (GSH) level in dose dependent manner. These results suggest that α-lipoic acid remove arsenic from arsenic-loaded isolated liver tissues of rat.
... Therefore, it is important to determine which is the active compound, ALA or DHLA, to exert various beneficial effects. It is known that ALA and DHLA do not show equivalent efficacy in several physiological activities (Muller and Menzel 1990;Lapenna et al. 2003;Coleman et al. 2006). The present study was performed to elucidate the differences in the cytotoxic activities of ALA and DHLA. ...
Alpha-lipoic acid has been shown to possess cancer-cell-killing activity via activation of the apoptosis pathway. In this study, the cytotoxic activities of alpha-lipoic and dihydro-alpha-lipoic acid were compared in HL-60 cells. The cell-killing activity of dihydro-alpha-lipoic acid was higher than that of alpha-lipoic acid. Both alpha-lipoic and dihydro-alpha-lipoic acid induced caspase-3 cleavage and internucleosomal DNA fragmentation in treated cells. On the other hand, apparent necrotic or late-stage apoptotic cell populations could be detected in dihydro-alpha-lipoic acid cells but not in those treated with alpha-lipoic acid. Moreover, dihydro-alpha-lipoic acid, but not alpha-lipoic acid, induced marked mitochondrial permeability transition. Antioxidants could not prevent dihydro-alpha-lipoic- or alpha-lipoic-acid-induced cell death. In addition, dihydro-alpha-lipoic and alpha-lipoic acid did not up-regulate cellular reactive oxygen level. These results indicated that dihydro-alpha-lipoic acid exerts more potent cytotoxicity than alpha-lipoic acid through different cytotoxic actions.
... Concentration of As 3+ was chosen based on prior studies of our group where 50 M As 3+ resulted in a minimal reduction in cell viability on HepG2 cells and still was able to induce a strong expression of stress genes and related proteins. Concentration of ALA was the same as used by Muller and Menzel (1990) which was able to protect rat hepatocytes from cadmium toxicity and also showed no signs of toxicity on HepG2 cells in previous experiments by our group. After exposure for 8 h, cells were washed twice with PBS and collected for analysis of MTT-metabolic activity, GSH levels, analysis of gene expression of several stress and antioxidant-related genes and Nrf2 induction. ...
Oxidative cell damage is a natural occurring phenomenon due to the aerobic conditions where cells are embedded; however, such injury can efficiently be controlled and repaired by the inherent antioxidants of the cell. When the oxidant/antioxidant balance is disrupted towards the former by any chemical, biological or physical insult a pathological state could be developed, therefore, an extensive list of compounds with proved or assumed antioxidants properties has largely been used for improving or restoring the health status. Pharmacological therapies as well as dietary or complementary therapies are continuously being investigated for the counteracting of the harmful or damaging effects of oxidation in cells or tissues. However, critical antioxidant levels are not always achieved at the target damaged cells by these approaches; on the other side, the expression of recombinant antioxidant genes specifically directed to the afflicted cell by gene delivery has shown remarkable results. In this review we summarize the literature focused on some of the current antioxidant molecular pharmacological strategies with particular emphasis to the gene transfer protocols involved in the treatment of oxidative stress-related disorders.
... Zinc is known to stabilize plasma and lysosomal membranes (Bettger & O'Dell, 1981). Cd 2+ (Muller & Menzel, 1990) and Hg 2+ ( Benov et al., 1990;Rungby & Ernst, 1992) induce events that lead to lipid peroxidation in cellular membranes. It is possible that mild peroxidation events that do not lead to cellular toxicity nevertheless interfere with LPS interactions important in signal transduction. ...
Metal salts can inhibit cell activity through direct toxicity to critical cellular molecules and structures. On the other hand, they can also change cell behaviour by inducing specific genes (including genes encoding members of the metallothionein [MT] gene family). Therefore, transition metals may affect cell functions either by acting as a toxin, or by transmitting or influencing signals controlling gene expression.
To explore the latter possibility, we measured the ability of low, non-toxic metal pretreatment to alter immune cell behaviour. We previously found that pretreatment of human monocytes with zinc induces metallothionein gene expression and alters their capacity to undergo a bacterial lipopolysaccharide-induced respiratory burst. We showed here that cadmium and mercury salts, at concentrations that exert no discernible toxicity, inhibit activation of human monocytic leukemia (THP-1) cells. CdCl2 1 μm, ZnCl2 20–40 μm or HgCl2 2 μm pretreatment for 20 h induced MT-2 mRNA and total MT protein accumulation and had no effect on proliferation potential or metabolic activity, but significantly inhibited the ability of subsequent lipopolysaccharide treatment to induce the oxidative burst, increased adhesion to plastic, and MT-2 and interleukin-1β (IL-1β) mRNA accumulation.
The phenomenon of metal-induced suppression of monocyte activation, at metal concentrations that have no effect on cell viability, has important implications for assessment of acceptable levels of human exposure to cadmium, zinc and mercury.
British Journal of Pharmacology (1997) 120, 797–806; doi:10.1038/sj.bjp.0700975
Glutathione (GSH) protects cells against oxidative stress. Redox modifiers induce GSH biosynthesis and recycling to maintain reduced environment inside cells. Cadmium (Cd²⁺) is a heavy metal that activates redox-sensitive transcriptional factors. The antioxidant α-lipoic acid (ALA) has shown to modulate GSH pathways. This study aimed to investigate de novo synthesis and recycling pathways for GSH balance by different Cd²⁺ concentrations and ALA in HepG2 cells. ALA activates Nrf2 pathway leading to GSH increment. Pre-treatment with 1 μM Cd²⁺ or ALA produces tolerance to 5 μM Cd²⁺ toxic effects. 5 μM Cd²⁺ exposure significantly augmented nuclear Nrf2, GSH and GCLC, GCLM, HMOX1, TNFα and IL-6 mRNA expression but not GSR, however these upsurges were significantly abrogated by ALA or 1 μM Cd²⁺ pre-treatments. Exposure to low Cd²⁺ concentration generate timely protective responses, similar to that elicited by ALA, maintaining a normal redox balance inside the cell due to GSH replenishment.
The antioxidant capacity of the deprotonated forms of lipoic and dihydrolipoic acids through their formation of complexes with copper has been theoretically studied. The relative stability of the various Cu(ii) complexes considered has been studied at the M06-2X/6-31++G(d, p) level of theory combined with the SMD continuum solvation model in water under physiological pH conditions. The most stable complexes of Cu(ii) are those formed with deprotonated dihydrolipoate when coordination involves the carboxylate group and one of the deprotonated thiol groups (in particular the primary one). The most thermodynamically stable Cu(ii) complex was found to have antioxidant capacity, since its presence can slow down by two orders the first step of the Haber-Weiss cycle (from 1.29 × 10⁸ M⁻¹ s⁻¹ to 1.33 × 10⁶ M⁻¹ s⁻¹) and reduce the potential damage caused by •OH radical formation.
Lipoic acid, the biomolecule of vital importance following glycolysis, shows diversity in its thiol-disulfide equilibria and also in its eight different protonation forms of the reduced molecule. In this paper, lipoic acid, lipoamide and their dihydro derivatives were studied to quantify their solubility, acid-base, and lipophilicity properties at a submolecular level. The acid-base properties are characterized in terms of 6 macroscopic, 12 microscopic protonation constants and 3 interactivity parameters. The species-specific basicities, the pH-dependent distribution of the microspecies and lipophilicity parameters are interpreted by means of various intramolecular effects, and contribute to understanding the antioxidant, chelate-forming and enzyme cofactor behavior of the molecules observed. This article is protected by copyright. All rights reserved.
Lipoic acid (LA) is an endogenous thiol which is reduced to dihydrolipoic acid (DHLA) in vivo. Both LA and DHLA act as antioxidants as do ascorbic acid (AA) and its oxidative product, dehydroascorbic acid (DHAA). It has been postulated that LA and DHLA interact directly with peroxy radicals in the cellular membrane and can recycle tocopheroxyl radicals back into tocopherol by a cascade mechanism involving the reduction of ascorbate. AA and DHAA have also been shown to have antitumor activity. However, very little is known about the cytotoxicity of DHLA and LA. In the present study, we compared the antitumor activity of DHLA and LA on the growth of murine leukaemia P388D1 cell line to AA and DHAA. DHLA was very lethal to the fast growing malignant cells even at a low concentration with ED50 0.4 μg/ml. In contrast, LA, AA and DHAA were non-toxic at lower concentrations, but toxicity increased with increasing concentrations. LA has an ED50 of 6.0 μg/ml whereas both AA and DHAA have an ED50 of 3.5 μg/ml. These results suggest that DHLA is considerably more cytotoxic than AA and DHAA.
Redox active molecules containing organoselenium or organotellurium groups catalyse the oxidation of cellular thiols by hydrogen peroxide and are currently being developed as therapeutic agents. Potentially these synthetic thiol peroxidase (TPx) mimics can protect cells from oxidative stress by catalysing the reduction of reactive oxygen species by the cellular thiol glutathione, an activity which mimics the function of the antioxidant enzyme glutathione peroxidase. Alternatively they can act as prooxidants by catalysing the oxidation of essential thiol species within the cell. However the structure activity relationships which determine the choice of thiol substrate, and hence the overall antioxidant or prooxidant outcome of drug administration, remain unknown. We report the first study that relates the pharmacological properties of TPx mimics with their solubility and catalytic activity using different thiol substrates. We used a series of structurally related compounds PhMCnH2n+1 (M=Se,Te; n=4-7) and investigated their ability to catalyse the oxidation of the cellular thiols glutathione and dihydrolipoic acid by hydrogen peroxide. The resulting rate constants (kobs) were then related to compound cytotoxicity and antioxidant versus prooxidant action in A549 cancer cells. The results show that the dihydrolipoic acid kobs values correlate with both cytotoxicity and prooxidant function. This enabled us to define a relationship, IC50=10+280e-5(DHLA kobs), which allows the prediction of TPx mimic cytotoxicity. In contrast, hydrophobicity and glutathione kobs were unrelated to the compounds’ redox pharmacology.
Building materials can be significant pollutant emission sources and can therefore be of public health importance. Transition elements, which form coloured ions, are utilized in making pigments which are used in various sections of the economy; electric lamps emit mercury, and various other household wares have been shown to consist of heavy metals other than lead. This chapter considers the heavy metals antimony, cadmium, chromium and mercury employed as building materials with respect to their history, structure, properties and uses. The biomonitoring, toxicological mechanisms and health effects of these metals as regards indoor pollution and remedial measures are discussed.
The aim of this study was to determine the effects of vitamin E, C, and α-lipoic acid additions to the feed rations on the serum glucose, lipid profile, and proteins in Japanese quails (Coturnix coturnix japonica) under heat stress. The serum levels of phosphorus, calcium, and liver enzymes were also measured. All groups were kept under 34oC during the hours between 0800 and 1700 and the temperature was 24°C during the rest part of the day. The control group (BS) was fed a normal ration without having any supplement, while the other groups had a supplementation of vitamin E (BSE), vitamin C (BSC), vitamin E+C (BSEC), and lipoic acid (BSLA). The serum glucose level was found to be lower in BSE, BSC, and BSLA groups compared to the BS group (P<0.05). The triacylglycerol (TAG) percentage was the lowest in the BSEC group (P<0.05). The diacylglycerol (DAG) percentage in the BSEC and BSLA groups was lower than the other groups (P<0.001). The cholesterol percentage in BSC, BSEC, and BSLA was higher than in the BS group (P<0.001). The polarlipid percentage was highest in the BSEC group while the lowest ones were in the BS and BSE groups. The highest level of total protein was in the BSEC group (P<0.05). The levels of albumin in the BS, BSC and BSLA groups were significantly higher than in the BSE group (P<0.05). The highest level of globulin was detected in the BSEC group while the lowest one was in the BSE group (P<0.001). The level of phosphorus was significantly high in the BSLA group (P<0.05), and the level of calcium did not differ among the groups. The levels of liver enzymes in the groups fed additives tended to decrease without having any statistical significance. In conclusion, vitamin E decreased the glucose and globulin and increased the albumin; vitamin C decreased the glucose; vitamin E+C decreased the TAG and DAG and increased the protein and globulin; the lipoic acid increased the glucose, DAG, and phosphorus.
An efficient, short and convenient asymmetric synthesis of (R)-(+)-lipoic acid in seven steps from chiral hydroxy aldehyde with 32.5% overall yield is described. Synthesis of S and R enantiomers of α-lipoic acid from cis-1,4-butene diol derived chiral lactone is reported with 34 % overall yield. The present synthesis involves use of simple reaction conditions making it a convenient synthesis.
Alpha-lipoic acid (α-lipoic acid) is a potent antioxidant compound that has been shown to possess anti-inflammatory effects. RAW 264.7 macrophages produce various inflammatory mediators such as nitric oxide, IL-1β, IL-6 and TNF-alpha upon activation with LPS ( Lipopolysaccharide) and IFNγ (interferon gamma). In this study, the effect of 12 synthetic indole α-lipoic acid derivatives on nitric oxide production and iNOS (inducible nitric oxide synthase) protein expression in LPS/IFNγ activated RAW 264.7 macrophages was determined. Cell proliferation, nitric oxide levels and iNOS protein expression were examined with thiazolyl blue tetrazolium blue test, griess assay and western blot, respectively. Our results showed that all of the indole α-lipoic acid derivatives showed significant inhibitory effects on nitric oxide production and iNOS protein levels (p < 0.05). The most active compounds were identified as compound I-4b, I-4e and II-3b. In conclusion, these indole α-lipoic acid derivatives may have the potential for treatment of inflammatory conditions related with high nitric oxide production. Copyright © 2015 John Wiley & Sons, Ltd.
Copyright © 2015 John Wiley & Sons, Ltd.
Cadmium (Cd) is a well-known human carcinogen and a potent nephrotoxin. The present study was carried to evaluate the protective effects of alpha-lipoic acid and melatonin against cadmium (Cd) induced oxidative stress to erythrocytes in rats. One hundred male albino rats were divided into five groups containing 20 rats each. Group I: (control) administered distilled water. Group II: (Cadmium exposed group) received cadmium chloride (4.4 mg kg-1 b.wt. of l/20th of LD50) orally and once per day over a period of 10 weeks. Group III: (Cadmium+Alpha-lipoic acid treated group) received cadmium chloride (4.4 mg kg-1 b.wt.) and treated daily with alpha-lipoic acid (54 mg kg-1 b.wt./i.p). Group IV: (Cadmium+Melatonin treated group) received cadmium chloride (4.4 mg kg-1 b.wt.) and treated daily with melatonin (10 mg kg-1 b.wt./orally). Group V: (Cadmium+alpha-lipoic acid and melatonin treated group). Heparinized blood used for Glucose-6-phosphate dehydrogenase and hemoglobin determination. Plasma used for determination of Alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST) and Gamma-glutamyltransferase (y-GT) activities and urea, creatinine, total cholesterol and phospholipids concentrations. Moreover, erythrocyte hemolysate were processed for the determination of L-Malondialdhyde (L-MDA), Catalase (CAT), Superoxide Dismutase (SOD), Glutathione-s-transferaseand (GST) and reduced Glutathione (GSH). Also, liver and kidney specimens were excised for histopathological examination and for cadmium residues determination. The obtained results revealed that, a significant increase in plasma ALT, AST and GGT activities, urea, creatinine, total cholesterol and phospholipids concentrations and erythrocyte L-MDA level, SOD activity,Liver and kidney cadmium residue were observed in cadmium intoxicated rats. However, administration of alpha-lipoic acid, melatonin and their combination in cadmium intoxicated rats exhibited a significant decreased in all mentioned parameters. On the other hand, a significant decreased in erythrocyte CAT, GST and G-6-PDH activities, GSH and hemoglobin concentrations were observed in cadmium intoxicated rats. Meanwhile, alpha-lipoic acid and melatonin administrations alone and in combination in cadmium intoxicated rats resulted in significant increase in all mentioned parameters. The histopathological studies in the liver and kidney of rats also supported that alpha-lipoic acid and melatonin markedly reduced the Cd induced pathological changes and preserved the normal histological architecture of the liver and kidney tissues.lt could be concluded that, the potential of alpha-lipoic acid and melatonin as a powerful agents and may be useful as an antioxidants in combating free radical-induced oxidative stress and tissue injury that is a result of cadmium toxicity. Also, these compounds have a protective antioxidant effect and could be also applicable as a cytoprotective against oxidative stress of tissue damage mediated by heavy metals intoxication.
Data presented on the tissue glutathione content in the liver and kidney of Gourami Trichogaster Leeri exposed for five weeks to sub lethal concentrations of Cadmium. The results indicate that Cadmium caused a marked elevation of glutathione concentrations in both liver and kidney tissue. Glutathione concentration in liver and kidney of Gourami exposed to 10 mg/L Cd were 350% and 400% of control level, respectively. The possibility of detecting environmental pollution by monitoring glutathione concentrations or the activity of glutathione dependent enzymes in fishes is discussed.
Heavy metals are known to cause oxidative deterioration of bio-molecules by initiating free radical mediated chain reaction resulting in lipid per-oxidation, protein oxidation and oxidation of nucleic acid like DNA and RNA. The development of effective dual functioning antioxidants, possessing both metal-chelating and free radical-scavenging properties should bring into play. Administration of natural and synthetic antioxidants like, quercetin, catechin, taurine, captopril, gallic acid, melatonin, N-acetyl cysteine, α- lipoic acid and others have been recognized in the disease prevention and clinical recovery against heavy metal intoxication. These antioxidants affect biological systems not only through direct quenching of free radicals but also via chelation of toxic metal(s). These antioxidants also, have the capacity to enhance cellular antioxidant defense mechanism by regenerating endogenous antioxidants, such as glutathione and vitamin C and E. They also influence cellular signaling and trigger redox sensitive regulatory pathways. The reactivity of antioxidants in protecting against heavy metal induced oxidative stress depends upon their structural properties, their partitioning abilities between hydrophilic and lipophilic environment and their hydrogen donation antioxidant properties. Herein, we review the structural, biochemical and pharmacological properties of selected antioxidants with particular reference to their ability to (i) chelate heavy metals from its complex (ii) ameliorate free radical (iii) terminate heavy metal induced free radical chain reaction (iv) regenerate endogenous antioxidants and, (v) excretion of metal without its redistribution.
Synthesis of α-lipoic acid has been achieved by a simple sequence of reactions. The synthesis highlights the use of α-chloroesters in a Reformatsky reaction. The intermediate keto acid is an intermediate from which both isomers of lipoic acid can be prepared.
Lipoic acid has been reported recently to be an effective antioxidant in biological systems. It may act in vivo through reduction to its dithiol form, dihydrolipoic acid. Using a dual Hg/Au electrode, and HPLC with electrochemical detection, a method was developed which allowed simultaneous measurement of lipoic acid and dihydrolipoic acid, at nanomolar levels. (RS)-α-Lipoic acid was added to human cells in tissue culture (Jurkat T-lymphocytes and primary neonatal diploid fibroblasts). Lipoic acid was converted rapidly by the cells to dihydrolipoic acid, which accumulated in the cell pellet. Monitored over a 2-hr interval, dihydrolipoic acid was released, and several-fold more dihydrolipoic acid could be found in the medium than in the pellet.
In cellular, tissue, and organismal systems, exogenously supplied α-lipoic acid (thioctic acid) has a variety of significant effects, including direct radical scavenging, redox modulation of cell metabolism, and potential to inhibit oxidatively-induced injury. Because reduction of lipoate to dihydrolipoate is a crucial step in many of these processes, we investigated mechanisms of its reduction. The mitochondrial NADH-dependent dihydrolipoamide dehydrogenase exhibits a marked preference for R(+)-lipoate, whereas NADPH-dependent glutathione reductase shows slightly greater activity toward the S(−)-lipoate stereoisomer. Rat liver mitochondria also reduced exogenous lipoic acid. The rate of reduction was stimulated by substrates which increased the NADH content of the mitochondria, and was inhibited by methoxyindole-2-carboxylic acid, a dihydrolipoamide dehydrogenase inhibitor. In rat liver cytosol, NADPH-dependent reduction was greater than NADH, and lipoate reduction was inhibited by glutathione disulfide. In rat heart, kidney, and brain whole cell-soluble fractions, NADH contributed more to reduction (70–90%) than NADPH, whereas with liver, NADH and NADPH were about equally active. An intact organ, the isolated perfused rat heart, reduced R-lipoate six to eight times more rapidly than S-lipoate, consistent with high mitochondrial dihydrolipoamide dehydrogenase activity and results with isolated cardiac mitochondria. On the other hand, erythrocytes, which lack mitochondria, somewhat more actively reduced S- than R-lipoate. These results demonstrate differing stereospecific reduction by intact cells and tissues. Thus, mechanisms of reduction of α-lipoate are highly tissue-specific and effects of exogenously supplied α-lipoate are determined by tissue glutathione reductase and dihydrolipoamide dehydrogenase activity. Copyright © 1996 Elsevier Science Inc.
Purpose
The aim of this study was to investigate whether the daily administration of α-lipoic acid (LA) during 4 weeks prevents the redox disturbance in red blood cells (RBC) described in diabetes
Methods
Multiple low-dose streptozotocin (STZ) diabetes was induced in rats by the administration of 40 mg/kg STZ intraperitoneally (i.p.) for 5 consecutive days. LA was applied at a dose of 10 mg/kg i.p. for 4 weeks, starting from the last day of STZ administration.
Results
The LA-treated diabetic rats exhibited a general systemic improvement, revealed as the near restoration of body weight and of essential biochemical parameters. The latter was displayed as decreased hyperglycemia, lower triglyceride levels and lower serum activities of alanine aminotransferases and aspartate aminotransferases that point to a general improvement of diabetes-linked organ “lesions”. The LA-treated diabetic rats also exhibited significant alleviation of oxidative stress, manifested as decreased lipid peroxidation and lower glycation levels of serum proteins and hemoglobin, while the RBC exhibited increased activities of antioxidant enzymes and elevated levels of reduced glutathione. In RBC, this was accompanied by decreased post-translational glycosylation by O-bound β-N-acetylglucosamine (O-GlcNAc) of the antioxidant enzymes superoxide dismutase and catalase and of heat shock proteins HSP70 and HSP90.
Conclusion
LA through its powerful antioxidant activity preserves the structural and functional integrity of RBC in diabetes. The RBC can then assume a more efficient role as the first line of systemic defense against diabetic complications arising from oxidative stress–induced damage of other tissues and organs.
The aim of this study was to see whether the taurine (TAU), alpha-lipoic acid (LA), curcumin (CUR), and N-acetylcysteine (NAC) protection against oxidative stress caused by heavy metals is owed to the metal-decreasing or antioxidative effect. In this context, liver and kidney tissues of common carp (Cyprinus carpio carpio L.) were exposed in vivo to model toxicants cadmium (Cd) and chromium (Cr). The tissues were dissected 96 h after intraperitoneal injection of the metals and antioxidant substances. Cd and Cr levels were determined in the liver using the ICP-OES, but we could not obtain enough kidney tissue to make the same measurements in the kidney. The enzymatic activities of SOD, CAT, and GPx, and the GSH redox status and lipid peroxidation levels were analyzed using spectrophotometric methods. Of all investigated antioxidants, only NAC decreased metal levels in the liver. Cd had little effect on oxidative stress parameters, while Cr showed a weak prooxidative effect. Cotreatment with TAU/LA/CUR/NAC and Cr significantly increased liver SOD activity. Chromium induced kidney SOD and CAT, but all antioxidants lowered CAT activity. Cadmium reduced liver and increased kidney GSSG. NAC increased liver GSH, but the increase did not correlate with decrease in Cd. Curcumin given with Cd increased kidney and decreased liver lipid peroxidation, whereas TAU with Cr increased lipid peroxidation in both tissues. N-Acetylcysteine was the most effective antioxidative agent, owing to its metal-decreasing function as well as to its effects on the GSH redox status. We believe that the investigated antioxidant substances which may have been involved in the reduction of Cr caused an increase in SOD activity and a decrease in CAT activity. Changes in the GSSG levels in both tissues might be an adaptive response to the prooxidative potential of Cd. Because of their respective tissue- and metal-dependent prooxidative effects, CUR and TAU deserve particular attention in regard to their use against metal toxicity, Cr in particular. © 2011 Wiley Periodicals, Inc. Environ Toxicol, 2011.
Chelation therapy occupies a central place in modern medicine and pharmacology, because continuous studies with laboratory animals and extensive clinical experience demonstrate that acute or chronic intoxications with a variety of metals can be considerable improved by administration of a suitable chelating agent. In this review the chemical characteristics, properties and uses of the most common chelating agents as well as those of some new and very promising agents of this type, are discussed. In the second part of the review the biological and biochemical impact of these agents, as well as their use for the treatment of some selected diseases and disorders, are also analyzed and discussed in detail.
Thesis (M. Med. Sc.)--University of Hong Kong, 2000. Includes bibliographical references (leaves 28-34).
Thioctic (lipoic) acid is used as a therapeutic agent in a variety of diseases in which enhanced free radical peroxidation of membrane phospholipids has been shown to be a characteristic feature. It was suggested that the antioxidant properties of thioctic acid and its reduced form, dihydrolipoic acid, are at least in part responsible for the therapeutic potential. The reported results on the antioxidant efficiency of thioctic and dihydrolipoic acids obtained in oxidation models with complex multicomponent initiation systems are controversial. In the present work we used relatively simple oxidation systems to study the antioxidant effects of dihydrolipoic and thioctic acids based on their interactions with: (1) peroxyl radicals which are essential for the initiation of lipid peroxidation, (2) chromanoxyl radicals of vitamin E, and (3) ascorbyl radicals of vitamin C, the two major lipid- and water-soluble antioxidants, respectively. We demonstrated that: (1) dihydrolipoic acid (but not thioctic acid) was an efficient direct scavenger of peroxyl radicals generated in the aqueous phase by the water-soluble azoinitiator 2,2'-azobis(2-amidinopropane)-dihydrochloride, and in liposomes or in microsomal membranes by the lipid-soluble azoinitiator 2,2'-azobis(2,4-dimethylvaleronitrile); (2) both dihydrolipoic acid and thioctic acid did not interact directly with chromanoxyl radicals of vitamin E (or its synthetic homologues) generated in liposomes or in the membranes by three different ways: UV-irradiation, peroxyl radicals of 2,2'-azobis(2,4-dimethylvaleronitrile), or peroxyl radicals of linolenic acid formed by the lipoxygenase-catalyzed oxidation; and (3) dihydrolipoic acid (but not thioctic acid) reduced ascorbyl radicals (and dehydroascorbate) generated in the course of ascorbate oxidation by chromanoxyl radicals. This interaction resulted in ascorbate-mediated dihydrolipoic acid-dependent reduction of the vitamin E chromanoxyl radicals, i.e. vitamin E recycling. We conclude that dihydrolipoic acid may act as a strong direct chain-breaking antioxidant and may enhance the antioxidant potency of other antioxidants (ascorbate and vitamin E) in both the aqueous and the hydrophobic membraneous phases.
Thioctic acid (TA) and its reduced form dihydrolipoic acid (DHLA) have recently gained some recognition as useful biological antioxidants. In particular, the ability of DHLA to inhibit lipid peroxidation has been reported. In the present study, the effects of TA and DHLA on reactive oxygen species (ROS) generated in the aqueous phase have been investigated. Xanthine plus xanthine oxidase-generated superoxide radicals (O2-), detected by electron spin resonance spectroscopy (ESR) using DMPO as a spin trap, were eliminated by DHLA but not by TA. The sulfhydryl content of DHLA, measured using Ellman's reagent decreased subsequent to the incubation with xanthine plus xanthine oxidase confirming the interaction between DHLA and O2-. An increase of hydrogen peroxide concentration accompanied the reaction between DHLA and O2-, suggesting the reduction of O2- by DHLA. Competition of O2- with epinephrine allowed us to estimate a second order kinetic constant of the reaction between O2- and DHLA, which was found to be a 3.3 x 10(5) M-1 s-1. On the other hand, the DMPO signal of hydroxyl radicals (HO.) generated by Fenton's reagent were eliminated by both TA and DHLA. Inhibition of the Fenton reaction by TA was confirmed by a chemiluminescence measurement using luminol as a probe for HO.. There was no electron transfer from Fe2+ to TA or from DHLA to Fe3+ detected by measuring the Fe(2+)-phenanthroline complex. DHLA did not potentiate the DMPO signal of HO. indicating no prooxidant activity of DHLA. These results suggest that both TA and DHLA possess antioxidant properties. In particular, DHLA is very effective as shown by its dual capability by eliminating both O2- and HO..
Rat hepatocyte suspensions were exposed to toxic concentrations of cadmium (Cd) in the presence and absence of unesterified alpha-tocopherol (T) or alpha-tocopheryl succinate (TS). The exogenous administration of TS completely protected hepatocytes from Cd-induced injury and lipid peroxidation. However, hepatocytes exposed to T were not protected from the toxic manifestations of cadmium even though this treatment resulted in a rapid marked accumulation of cellular T. The rate of cadmium uptake by hepatocytes was not significantly altered by exogenous TS or T treatment. These studies indicate that TS cytoprotection against Cd toxicity results not from alterations in Cd uptake or the accumulation of T but rather from the cellular presence of the intact TS molecule. The data also indicate that the depletion of cellular T is not the critical cellular event that is responsible for Cd-induced injury. Instead it appears that TS possess unique cytoprotective properties that intervene in the critical cellular events that lead to Cd toxicity. Thus, TS administration represents a promising new strategy for the mechanistic study and prevention of tissue damage resulting from Cd exposure.
alpha-Lipoic acid, which plays an essential role in mitochondrial dehydrogenase reactions, has recently gained considerable attention as an antioxidant. Lipoate, or its reduced form, dihydrolipoate, reacts with reactive oxygen species such as superoxide radicals, hydroxyl radicals, hypochlorous acid, peroxyl radicals, and singlet oxygen. It also protects membranes by interacting with vitamin C and glutathione, which may in turn recycle vitamin E. In addition to its antioxidant activities, dihydrolipoate may exert prooxidant actions through reduction of iron. alpha-Lipoic acid administration has been shown to be beneficial in a number of oxidative stress models such as ischemia-reperfusion injury, diabetes (both alpha-lipoic acid and dihydrolipoic acid exhibit hydrophobic binding to proteins such as albumin, which can prevent glycation reactions), cataract formation, HIV activation, neurodegeneration, and radiation injury. Furthermore, lipoate can function as a redox regulator of proteins such as myoglobin, prolactin, thioredoxin and NF-kappa B transcription factor. We review the properties of lipoate in terms of (1) reactions with reactive oxygen species; (2) interactions with other antioxidants; (3) beneficial effects in oxidative stress models or clinical conditions.
This chapter discusses the structure–antioxidant activity relationships of dihydrolipoic acid (DHLA). α-Lipoic acid/DHLA is effective as a therapeutic agent. In many cases, the effects of α-lipoic acid appear to be linked to its ability to influence oxidative stress-mediated processes. For example, α-lipoic acid dietary supplementation successfully prevents myocardial damage induced by ischemia-reperfusion; α-lipoic acid prevents macromolecular alterations induced by high concentrations of glucose; preincubation of cultured human T cells with α-lipoic acid inhibits activation of NF-κB, a transcription factor responsible for the activation of human immunodeficiency virus (HIV). Such findings on the effects of α-lipoic acid may relate to the antioxidant properties of DHLA, and thus, the development of DHLA-based antioxidants may have significant clinical ramifications. Success in such a strategy may require a more comprehensive approach based on the structure–function relationships of a compound, involving (1) determination of structure–activity relationships, (2) application of concepts derived from structural studies in cellular systems to elucidate the mechanism of action, and (3) verification of information in intact animals.
A selective and sensitive gas chromatographic method for the analysis of lipoic acid in biological samples has been developed. After base hydrolysis of the sample, the liberated lipoic acid was converted into its S,S-diethoxycarbonyl methyl ester derivative and measured by gas chromatography using a DB-210 capillary column and a flame photometric detector. The calibration curve was linear in the range 20-500 ng, and the detection limit was ca. 50 pg injected. The best hydrolysis conditions for the biological samples were obtained by using 2 M potassium hydroxide containing 4% bovine serum albumin at 110 degrees C for 3 h. Using this method, lipoic acid in the hydrolysate could be selectively determined without any interference from matrix substances. Analytical results for the determination of lipoic acid in the mouse tissue and bacterial cell samples are presented.
Uptake of [35S]lipoate was studied in perfused rat liver and in isolated rat hepatocytes. During single-pass perfusion of [35S]lipoate about 30% of the radioactivity is retained in the liver. A substantial amount of 5,5'-dithiobis(2-nitrobenzoic acid)-reactive material appears in the effluent perfusate, while hepatic efflux of GSH is unchanged. The hepatic uptake of lipoate, the release of thiols, and also the biliary excretion of 35S-labeled compounds are suppressed by octanoate. In isolated hepatocytes the uptake of lipoate follows saturation kinetics showing a Km value of 38 microM and a Vmax of 180 pmol/mg X 10 s. The uptake is temperature-dependent; from the Arrhenius plot an activation energy of 14.8 kcal/mol at 20 microM lipoate is calculated. At high concentrations of lipoate (above 75 microM) a nonsaturable uptake component becomes predominant. Lipoate uptake is selectively inhibited by medium-chain fatty acids. Only slight inhibition is seen in the presence of long-chain fatty acids, and there is no inhibition with acetate or lactate. Substantial inhibition is also observed with acetylsalicylic acid, but not with taurocholate, bromosulfophthalein or biotin. Lipoate uptake can be inhibited by high concentrations of phloretin (200 microM) and is rather insensitive to 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (200 microM). The results indicate that hepatic uptake of lipoate at physiological concentrations is largely carrier-mediated.
A 55-year-old woman suffered acute hepatic necrosis following ingestion of Amanita virosa mushrooms. Successful treatment included the use of thioctic acid. A discussion of Amanita poisoning and the therapeutic modalities available is presented.
An der isoliert perfundierten Rattenleber ist der Zusammenbruch der Kationengradienten (K+, Na+) das Primrereignis der Phalloidinvergiftung. Thioctsure hemmt, noch einige Minuten nach der Zugabe von Phalloidin verabfolgt, den Austritt von K-Ionen und Kathepsin aus der Leberzelle.In the perfused rat liver simultaneous release of potassium and penetrations of sodium is the first effect seen in Phalloidin intoxication. Lipoic acid given before or some minutes after Phalloidin protects the liver cell membrane against the above effect of the toxin.
Although various theories have been proposed to account for the mechanism of cadmium-induced cellular injury, none have received strong, direct support from experimental data. An interference with sulfhydryl groups is one of the proposed mechanisms. The ability of reduced glutathione (GSH) to protect hepatocytes from the toxic effects of cadmium has been investigated. When added just prior to cadmium, GSH has a pronounced protective effect, while an additional 15 min after cadmium resulted in partial protection against the ensuing loss of cellular potassium ion. The protection against loss of aspartate aminotransferase into the medium is pronounced, regardless of whether addition of GSH is at zero time or 15 min after cadmium. Addition of the GSH at either time resulted in reduced levels of cadmium associated with the hepatocytes, and may well account for these protective properties of GSH. Protein-bound sulfhydryl groups showed a small decrease in response to cadmium but only after cell injury had been initiated. These data allow the conclusion that cadmium does not exert its cytotoxic effects by simple, single irreversible interaction with cellular thiol groups. Furthermore, as the added GSH remains extracellular, these data indicate that protection can be afforded at an extracellular level even after intracellular exposure to toxic concentrations of cadmium.
Cadmium (Cd) (10–100 μM) decreased the ATP/ADP ratio and enhanced lipid peroxidation (LPO) (measured as thiobarbituric acid reactants) in incubated rat hepatocytes. Analysis of the subcellular distribution of Cd indicated its preferential attachment to the inner membranes of mitochondria. Incubation of isolated mitochondria with 0.005–0.05 μM Cd resulted in increased formation of formazans from nitroblue tetrazolium salts, indicating enhanced membrane permeability to succinate. These Cd-concentrations also diminished mitochondrial ATP. LPO in mitochondria strongly increased only after Cd-exposures above 1 μM Cd. Similarly, in Cd-treated hepatocytes decreases in ATP/ADP ratios corresponded to increases in LPO stimulation only at 30 and 60 min but not at 15 min of incubation when ATP/ADP ratios were already affected. Moreover, neither hepatocellular ATP/ADP decrease nor mitochondrial formazan formation due to Cd were prevented by (+)-cyanidanol-3, an effective inhibitor of Cd-induced LPO. These data suggest that even low Cd-concentrations in the hepatocyte disturb the integrity of its mitochondrial membranes concomitantly impairing the hepatocellular energy supply. LPO, only observed at higher Cd-concentrations, is not responsible for these adverse Cd-effects.
Net cadmium uptake in the isolated perfused rat iiver was half-maximal at 5 μM, and the maximal rate of uptake was 22 nmoles/min per gram liver wet weight. Uptake was augmented when a permeable thiol, dithioerythritol, was infused, whereas it was restricted when glutathione as a nonpermeable thiol or also when bovine serum albumin were infused. The ratio of extra cadmium taken up versus dithioerythritol added was 1:2. Uptake of cadmium was insensitive to anoxia or to the infusion of cyanide. Biliary cadmium release in the perfused liver was not augmented by dithioerythritol but was rather suppressed, whereas bile flow or the release of added 3H-taurocholate were unaffected.
This working paper summarizes the known ultrastructural and biochemical effects of lead, mercury, cadmium, and arsenic on subcellular organelle systems following in vivo administration. Documented metal-induced alterations in nuclear, mitochondrial, microsomal, and lysosomal functions are discussed in relation to their potential impact on cellular responses to other environmental agents. Each of the above elements has been found to interfere with normal cellular replication and genetic processes. Mitochondrial swelling and depression of respiratory function are discussed in relation to known metal-specific perturbations of mitochondrial heme biosynthetic pathway enzymes. Inhibition of microsomal enzyme activities and protein synthesis by lead and mercury is compared to the apparent absence of such effects following arsenic or cadmium exposure. Lysosomal uptake of all the metals is documented, but biochemical alterations in these structures have been reported for only mercury and cadmium. It is concluded that these toxic metals are capable of interacting with, and biochemically altering major cellular systems at dose levels below those required to produce signs of overt metal toxicity. The impact of these effects on cellular response to other metals and xenobiotics in complex exposure situations is presently unknown, and further research is urgently needed in this area.
Effects of cadmium (Cd) in vivo and in vitro were studied in the absence of enhanced metallothionein (MT) production and overt Cd toxicity. Such a condition was established by extended oral exposure of male rats of 0.2 μmol Cd/kg and by incubation of isolated hepatocytes with up to 25 μM for 30 min. Subsequently, mitochondrial and extramitochondrial responses to Cd were recorded. Cadmium diminished the activity of cytochrome c oxidase (CYT C OX) by 50% in vivo and by 35% in vitro. In hepatocytes, this was accompanied by increased Cd and decreased protoheme (PrH) in mitochondria. Extramitochondrial PrH and cytochrome P 450 were not significantly altered. In hepatocytes from phenobarbitone pretreated rats, 25 μM Cd decreased CYT C OX but not mitochondrial PrH. Moreover, simultaneous incubation of hepatocytes with 25 μM Cd and either 2.5 mM dithiothreitol or 5 mM reduced glutathione diminished cellular and mitochondrial Cd and prevented the decrease in CYT C OX but not that in PrH. In contrast, coincubation with either 250 μM
l-buthionine-sulfoximine or diethylmaleate, which did not alter Cd uptake, prevented the decrease in PrH but not that in CYT C OX owing to Cd. These results show that Cd exerts mitochondrial alterations in vivo and in vitro in the absence of enhanced MT production. Moreover, Cd effects on CYT C OX and PrH do not seem to be firmly linked.
The antioxidant effect of dihydrolipoate and lipoate was examined in microsomal fractions obtained from normal and alpha-tocopherol-deficient animals after initiation of lipid peroxidation with an NADPH/iron/ADP system. Dihydrolipoate prolonged the lag phase before the onset of low-level chemiluminescence and before the rapid accumulation of thiobarbituric acid-reactive substances in normal but not in vitamin E-deficient microsomes. Lipoate did not show such an antioxidant effect. It is concluded that the dihydrolipoate-mediated protection against lipid peroxidation by prolonging the lag phase is dependent on alpha-tocopherol. Likewise, dihydrolipoate prolonged the lag phase before the onset of the rapid loss of vitamin E during lipid peroxidation. Dihydrolipoate, like other biological thiols such as GSH, also affects the peroxidative process after the lag period. The effects included a smaller slope of the chemiluminescence increase, a lower maximal level of chemiluminescence, a slower loss of alpha-tocopherol and a slower accumulation, but unchanged maximal levels, of thiobarbituric acid-reactive substances. The biological significance may be most prominent in the mitochondrial matrix space, where lipoamide-containing ketoacid dehydrogenases are located. A potential pharmacological use of this biological dithiol in conditions associated with oxidative stress could be based on the antioxidant activity of dihydrolipoate.
The suitability of DL-alpha-lipoic acid (LA) to serve as an antidote in cadmium (Cd) toxicity in rat hepatocytes was investigated. Isolated hepatocytes were exposed to 200 and 450 microM Cd in the presence of 0.2, 1.0 and 5.0 mM LA, respectively. After 30 min of incubation various criteria of cell viability were monitored. Lipoic acid markedly diminished Cd uptake. Concomitantly, Cd-induced membrane injury, as reflected by the leakage of aspartate aminotransferase and sorbitol dehydrogenase (SDH) was decreased. Moreover, LA protected against intracellular toxic responses to Cd, such as a decrease in cellular SDH activity, a decrease in cellular acid soluble thiols, especially in total glutathione, a decrease in cellular urea and an increase in thiobarbituric acid (TBA) reactants, as a measure of lipid peroxidation. Most protective effects were seen in hepatocytes challenged with the lower Cd concentration and coincubated with 5 mM LA. In contrast, at 450 microM Cd even the highest LA concentration applied either did only reverse Cd-effects incompletely (SDH-response, TBA-reactants) or did not protect at all (Cd uptake, enzyme leakage, loss of glutathione). The data indicate that DL-alpha-lipoic acid serves as a protective tool against Cd-induced membrane damage and cell dysfunction in hepatocytes. This stands as long as Cd exposure is low enough to permit interaction with LA prior to interaction with cell structures.
Experimental modulation of cellular glutathione levels has been used to explore the role of glutathione in cadmium toxicity. Mice treated with buthionine sulfoximine [an effective irreversible inhibitor of gamma-glutamylcysteine synthetase (EC 6.3.2.2) that decreases cellular levels of glutathione markedly] were sensitized to the toxic effects of CdCl2. Mice pretreated with a sublethal dose of Cd2+ to induce metallothionein synthesis were not sensitized to Cd2+ by buthionine sulfoximine. Mice sensitized to Cd2+ by buthionine sulfoximine were protected against a lethal dose of Cd2+ by glutathione mono isopropyl ester (L-gamma-glutamyl-L-cysteinylglycylisopropyl ester), but not by glutathione. These results are in accord with studies that showed that glutathione mono esters (in contrast to glutathione) are efficiently transported into cells and converted intracellularly to glutathione. The findings indicate that intracellular glutathione functions in protection against Cd2+ toxicity, and that this tripeptide provides a first line of defense against Cd2+ before induction of metallothionein synthesis occurs. The experimental approach used here in which cellular levels of glutathione are decreased or increased seems applicable to investigation of other types of metal toxicity and of other glutathione-dependent biological phenomena.
The effects of low-level cadmium (Cd) administration to rats on animal health, liver and kidney thiols, metallothionein, and glutathione reductase (GSSG reductase) and their modulation by cysteine (as a possible protector) and diethyl maleate (as a possible potentiator) have been investigated. Male Sprague-Dawley rats were treated with sodium or Cd acetate (25 micrograms Cd/kg) orally five times a week for 6 weeks. A second group of animals received cysteine (500 mg/kg; po) before each gavage while a third group received diethyl maleate (DEM) (0.85 mg/kg; ip) in addition to sodium or Cd acetate. When rats were treated with cadmium alone neither weight gain nor serum parameters indicative of hepato- or nephro-toxicity were affected. However, acid-soluble thiols, primarily glutathione, were decreased by about 25% in liver only. A tendency to a decrease in hepatic protein thiols was also noted. No changes were observed for hepatic or renal metallothionein in response to this low level of cadmium administration alone or in combination with the other treatments. Animals receiving cysteine, either alone or with cadmium, showed decreased body weight gain, but no change in serum parameters. Acid-soluble thiols in liver were lower in cysteine-treated rats (24%) and cysteine + Cd (33%) while kidney thiols were unaffected. Administration of DEM alone or with Cd did not cause any alteration in body weight gain. When given DEM + Cd, however, an increase in serum bilirubin was observed, which suggests interference with hepatobiliary function. Acid-soluble thiols were decreased by DEM alone (45%) and DEM + Cd (51%) in liver while renal thiols showed no change. Our data indicate that low-level Cd gavage decreases hepatic cellular thiols but not those of kidney. Cysteine gavage does not protect from the Cd-related effect. Indeed, cysteine itself was found to reduce acid-soluble thiols under the experimental conditions. This was observed only in liver, as was the decrease in thiols due to DEM treatment. DEM administration together with Cd resulted in signs of liver toxicity. There is no indication that inhibition of GSSG reductase by Cd might be involved in the thiol-decreasing effect of short-term repeated low-level gavage of Cd to rats.
Reduced glutathione (GSH) delays microsomal lipid peroxidation via the reduction of vitamin E radicals, which is catalyzed by a free radical reductase (Haenen, G.R.M.M. et al. (1987) Arch. Biochem. Biophys. 259, 449-456). Lipoic acid exerts its therapeutic effect in pathologies in which free radicals are involved. We investigated the interplay between lipoic acid and glutathione in microsomal Fe2+ (10 microM)/ascorbate (0.2 mM)-induced lipid peroxidation. Neither reduced nor oxidized lipoic acid (0.5 mM) displayed protection against microsomal lipid peroxidation, measured as thiobarbituric acid-reactive material. Reduced lipoic acid even had a pro-oxidant activity, which is probably due to reduction of Fe3+. Notably, protection against lipid peroxidation was afforded by the combination of oxidized glutathione (GSSG) and reduced lipoic acid. It is shown that this effect can be ascribed completely to reduction of GSSG to GSH by reduced lipoic acid. This may provide a rationale for the therapeutic effectiveness of lipoic acid.
The biochemical mechanism involved in cadmium-induced cellular injury remains to be elucidated. Various theories have been proposed to account for this action, one of which is an interaction with reduced sulfhydryl (SH) groups of membranes. The ability of a known SH group reducing agent, dithiothreitol (DTT), to interact with cadmium-induced damage to isolated hepatocytes was investigated. Cadmium chloride produced a cytotoxic response similar to that previously demonstrated. When added just prior to cadmium chloride, DTT was found to protect against the metal-induced injury. If the cells were first exposed to cadmium for 15 min before DTT was added, it was found that the cells could be rescued from the ensuing expression of toxicity due to cadmium. This result was regardless of the fact that by 15 min the cells had accumulated a quantity of cadmium that was associated with cell injury. Furthermore, DTT did not exert its action by decreasing the amount of cadmium accumulation by the cells. Under some experimental conditions, an increased cadmium uptake in the presence of DTT was observed. While it is not clear as to whether the chelating or thiol reductive properties of DTT are of prime importance in the demonstrated protection, the data show that toxicity is not due to a simple, single irreversible interaction between cadmium and membrane SH groups.
A method for the analysis of nanogram quantities of glutathione has been developed which is based on the catalytic action of GSH or GSSG in the reduction of Ellman reagent (DTNB) by a mixture of TPNH and yeast glutathione reductase. Unlike previous methods of analysis the procedure described here effectively measures the total glutathione (GSH + GSSG) content of unknown mixtures and is not subject to appreciable interference by the presence of other thiol components. It is suggested that the catalytic action of glutathione in this system resides in the continual enzymic regeneration of GSH, present initially or formed enzymically from GSSG, following its interaction with the sulfhydryl reagent.The sensitivity of the method is such as to permit the determination of total glutathione in extracellular tissue fluids such as plasma, saliva, and urine normally containing very low levels of this material, essentially without pretreatment of the sample. The same is true for glutathione determinations of whole blood, in which the preliminary procedure is confined to the preparation of a 1:100 hemolyzate from as little as 10 μl of sample.Following published procedures, the pretreatment of tissue extracts with NEM to form an enzymically inactive complex with free GSH allowed the determination of the low levels of oxidized glutathione normally present therein. The use of the foregoing analytical method in the determination of total and oxidized glutathione contents of rat blood, kidney, and liver gave values in good agreement with those obtained by previous investigators.
A review is presented of the chemical and biochemical effects of mercury, acadmium and lead. Similarities and diversities are emphasized and the means available to identify their biochemical sites of action are discussed. Toxic effects and alterations in enzyme activity are described. 551 references.
A randomised double-blind trial of thioctic acid (alpha-lipoic acid), 300 mg/day versus placebo was carried out in 40 patients with pre-cirrhotic alcohol-related liver disease over a six month period. Twenty patients received the active drug and 20 placebo. Twenty-two of the 40 patients (55%) abstained from alcohol and showed significant improvements (p less than 0.01) in mean values for serum aspartate transaminase, serum glutamyl transpeptidase, and mean corpuscular volume. Seventeen of the 22 (77%) showed overall histological improvement on liver biopsy. The remaining 18 patients (45%) continued to drink but significantly reduced their mean daily alcohol intake (p less than 0.001). No significant changes occurred in their laboratory indices, but five of the 18 (28%) showed overall histological improvement. Changes occurred irrespective of treatment with thioctic acid, which suggested that, over six months, this drug did not influence the course of alcohol-related liver disease.
The toxic effects of cadmium and other selected divalent cations are presumed to be related to specific chemical and physical characteristics of the ion. The chemistry of cadmium and metal ions in general is reviewed from the viewpoint of such relevant properties as ion polarizability, electronic structure, and the hard-soft characteristics. The softness of metal ions is seen as a useful single parameter to correlate with the affinity for nucleic acids and proteins and with toxic effects. The effects of cadmium on nucleic acids and proteins are examined for a number of specific cases to illustrate the variety of interactions that are well recognized and to demonstrate the utility of soft metal ions as reagents and probes for examining the relationship of structure and function in these macromolecules.
Various mechanisms, including increases in lipid peroxidation, have been proposed to account for metal-induced cellular injury. By comparing several metals in the same cell population, it is possible to determine whether a correlation exists between ability to produce cell injury and ability to alter parameters pertaining to a particular mechanism. Of particular interest in this study was the relation between metal-induced cytotoxicity and increases in lipid peroxidation. The effects of Cr, Mn, Zn, Ni, Pb, Se, V, Fe, Cd, Hg, Cu, at final concentrations of 1-1000 microM, on the viability of isolated hepatocytes were therefore examined by assessing the loss of intracellular K+ and aspartate aminotransferase (AST). Simultaneously, the ability of the metals to induce lipid peroxidation, as measured by an increase in thiobarbituric acid (TBA) reactants, was assessed. Hg and Cu required the lowest concentration to produce cellular injury, while Cd produced less dramatic changes in cell viability and Fe at 1000 microM produced only a small decrease in intracellular K+. The largest absolute increases in lipid peroxidation were found in the presence of V, followed by Fe and Hg, with Cd and Se causing the smallest increase in TBA reactants. These observations suggest that the lipid peroxidation associated with Cd and Hg is not necessarily responsible for the loss of cell viability induced by these two metals.
The incubation of isolated hepatocytes with 10-100 microM cadmium (Cd) decreased the ATP/ADP-ratio, the ATP/AMP-ratio and the adenylate energy charge and enhanced the lactate/pyruvate-ratio in a time- and concentration-dependent manner. Likewise, the cellular oxygen-consumption was decreased at early incubation times, when the cell membrane was still intact, as judged by the Trypan Blue-exclusion test. Similarly, Cd-concentrations above 0.3 nmol/mg mitochondrial protein inhibited the succinate- and malate/pyruvate-stimulated respiration of isolated mitochondria. This critical concentration was also reached in mitochondria of hepatocytes, when exposed to 25 microM Cd for less than 30 min. We therefore suggest that the inhibition of cellular respiration and the breakdown of cellular energy generation is mediated by a severe disturbance of mitochondrial respiratory functions due to Cd. This effect seems to be an early event in Cd-toxicity in isolated hepatocytes.
A 55-year-old woman suffered acute hepatic necorsis following ingestion of Amanita virosa mushrooms. Successful treatment included the use of thioctic acid. A discussion of Amanita poisoning an the therapeutic modalities available is presented.
Thioctic acid markedly increases the sulfhydryl and sulphide content of bile. This probably reflects the reduction of thioctic acid in the liver, followed by biliary excretion of a reduced derivative. The total biliary excretion of methyl mercury was not increased. Thionalide markedly inhibits biliary excretion of methyl mercury. Simultaneously, the sulfhydryl and sulphide content of bile decreases. This is probably caused by the conjugation of thionalide to glutathione in the liver, thereby blocking the biliary excretion of methyl mercury. Hexadecylmercaptoacetate increases the biliary content of methyl mercury moderately after a temporary decrease, whereas biliary sulfhydryl and sulphide concentrations were unchanged. Octadecylmercaptoacetate does not change the biliary content of methyl mercury, sulfhydryl and sulphides significantly. Smaller parts of hexadecylmercaptoacetate, octadecylmercaptoacetate and thionalide seemed to be excreted as such in bile. These results indicate that methyl mercury cannot be transported from liver to bile as complexed to the sulphides thioctic acid, thionalide, hexa- and octadecylmercaptoacetate.
From a study of acute heavy-metal toxicity it has been found that dl-α-lipoic acid (a) is effective for the prevention and reversal of arsenic intoxication in mice and dogs; (b) effectively prevents mercury intoxication in mice provided a sufficiently large excess is used; (c) prevents gold intoxication in mice only under very specific conditions; and (d) fails to protect mice against a lethal dose of lead.3-(6-Carboxyhexyl)-1,3-dithiolane was as effective as dl-α-lipoic acid in preventing arsenic intoxication in mice.
1.1. Dihydrolipoic acid readily forms chelates with a number of divalent heavy metals; these differ in stability to acid and other properties.2.2. The Co2+-dihydrolipoic acid chelate is oxidized immediately in air with the uptake of 1.5 atoms oxygen per mole chelate. Oxidation is accompanied by an increase in the molecular extinction coefficient from ϵ480mμ = 5.12·103 to ϵ510mμ = 1.45·104 cm2/mmole. Biologically active derivatives of lipoic acid cannot be recovered from the oxidized complex.3.3. Comparative studies with model compounds indicate that both sulphydryl groups of dihydrolipoic acid are involved in the chelation of Co2+, and presumably certain other cations.4.4. Spectrophotometric measurements, chemical analysis and electrometric titration show that in dilute solution dihydrolipoic acid and Co2+ combine initially in a 2:1 ration, the combination being accompanied by the liberation of one hydrogen ion per mole ligand.5.5. Chelates of dihydrolipoic acid with Cu2+ and Ni2+ also undergo oxidation at rates which, whilst similar to one another, are greater than that exhibited by dihydrolipoic acid alone, but considerably less than that of the Co2+ chelate. Oxidation of the Ni2+ chelate is not accompanied by any change in absorption spectrum.6.6. Chelates of dihydrolipoic acid with Zn2+ and Cd2+ are stable in air and exhibit no oxygen uptake. These chelates in contrast to those of Co2+ and Cu2+, dissociate in acid solution to yield the dithiol and essentially quantitative recovery of biological activity.7.7. It is suggested that the senstivity of the Co2+-dihydrolipoic acid chelate to oxidation may be of significance with regard to the inactivation by Co2+ of biological systems that are dependent upon the reversible interconversion of lipoic acid and dihydrolipoic acid.
Since 1922 when Wu proposed the use of the Folin phenol reagent for
the measurement of proteins (l), a number of modified analytical pro-
cedures ut.ilizing this reagent have been reported for the determination
of proteins in serum (2-G), in antigen-antibody precipitates (7-9), and
in insulin (10).
Although the reagent would seem to be recommended by its great sen-
sitivity and the simplicity of procedure possible with its use, it has not
found great favor for general biochemical purposes.
In the belief that this reagent, nevertheless, has considerable merit for
certain application, but that its peculiarities and limitations need to be
understood for its fullest exploitation, it has been studied with regard t.o
effects of variations in pH, time of reaction, and concentration of react-
ants, permissible levels of reagents commonly used in handling proteins,
and interfering subst.ances. Procedures are described for measuring pro-
tein in solution or after precipitation wit,h acids or other agents, and for
the determination of as little as 0.2 y of protein.
Detoxyfying effect of thioctic acid on mercury poisoning
- Ueda