The effect of ifosfamide and its metabolites on intracellular glutathione levels in vitro and in vivo

The effect of ifosfamide and its metabolites on intracellular levels of glutathione in P388 cells in vitro has been studied. It is demonstrated that glutathione depletion occurs only in the presence of 4-hydroperoxyifosfamide and chloroacetaldehyde. In contrast isophosphoramide mustard had no effect on glutathione levels in intact cells. The concentration of 4-hydroperoxyifosfamide required to reduce glutathione levels by 50% was approximately 1 mM and this represents a concentration far in excess of that achievable in patients receiving the drug. However the concentration of chloroacetaldehyde (approximately 100 microM) required to reduce intracellular levels of glutathione to a similar extent is attained in patients receiving ifosfamide. The glutathione levels in lymphocytes isolated from a patient undergoing an eight hour infusion of ifosfamide showed a marked decrease to about 30% of their original value. We conclude that ifosfamide causes glutathione depletion in vivo and the majority of this can be accounted for by the production of chloroacetaldehyde.     

Inhibition of adenosine diphosphate-induced platelet aggregation by alpha-lipoic acid and dihydroquercetin in vitro

Objectives:

To investigate the antiplatelet activity of alpha-lipoic acid (α-LA) and dihydroquercetin (DHQ).

Materials and

Methods: Antiplatelet activity of the α-LA and DHQ was evaluated in rich platelet plasma of rat. The platelet aggregation was induced by adenosine diphosphate (ADP) in concentration of 4 × 10^-5 M.

Results:

α-LA and DHQ inhibited platelet aggregation in concentration-dependent manner. The antiplatelet activity of α-LA was more pronounced than DHQ. DHQ also increased the antiplatelet activity of α-LA by 1.4 times.

Conclusion:

Combined simultaneous use of α-LA and DHQ possessed the high antiplatelet activity, and DHQ potentiated the activity of α-LA.KEY WORDS: Alpha-lipoic acid, antioxidants, antiplatelet activity, dihydroquercetin, platelet aggregation     

α-硫辛酸对2型糖尿病患者氧化应激及尿白蛋白排泄的影响

目的观察α-硫辛酸(ALA)对2型糖尿病患者氧化应激的影响及其肾脏保护作用。方法选择36例2型糖尿病患者为观察组,在糖尿病治疗的基础上加用ALA,并选择同期健康体检的30例作为对照组。6个月后,观察治疗前后空腹血糖(FBG)、糖化血红蛋白(HbA1c)、血清丙二醛(MDA)、超氧化物歧化酶(SOD)及尿白蛋白(ALB)水平变化。结果①糖尿病组血清MDA、UACR(Ualb/Ucr)升高,血清SOD活性降低,与对照组比较差异有统计学意义(P<0.01)。②ALA治疗6个月后,观察组FBG和HbA1c无明显变化,血清MDA、UACR降低,血清SOD活性明显升高,与治疗前相比较差异有统计学意义(P<0.01)。③UACR与血清MDA水平显著正相关(r=0.867,P<0.01),与血清SOD水平明显负相关(r=-0.636,P<0.01)。结论α-硫辛酸可以减轻2型糖尿病患者尿白蛋白排泄,该作用可能与其减轻体内氧化应激作用有关。     

New metabolic pathways of alpha-lipoic acid.

The excretion and biotransformation of rac-alpha-lipoic acid (LA), which is used for the symptomatic treatment of diabetic polyneuropathy, were investigated following single oral dosing of [(14)C]LA to mice (30 mg/kg), rats (30 mg/kg), dogs (10 mg/kg), and unlabeled LA to humans (600 mg). More than 80% of the radioactivity given was renally excreted. Metabolite profiles obtained by radiometric high-performance liquid chromatography revealed that LA was extensively metabolized irrespective of the species. Based on a new on-line liquid chromatography/tandem mass spectroscopy assay developed for negative ions, LA and a total of 12 metabolites were identified. Mitochondrial beta-oxidation played the paramount role in the metabolism of LA. Simultaneously, the circulating metabolites were subjected to reduction of the 1,2-dithiolane ring and subsequent S-methylation. In addition, evidence is given for the first time that the methyl sulfides formed were partly oxidized to give sulfoxides, predominantly in dogs. The disulfoxide of 2,4-bismethylmercapto-butanoic acid, the most polar metabolite identified, was the major metabolite in dogs. Furthermore, new data are presented that suggest conjugation with glycine occurred as a separate metabolic pathway in competition with beta-oxidation, predominantly in mice.     

Effect of beta-lactam antibiotics on hepatocellular glutathione levels in vitro and in vivo

Incubation of isolated rat hepatocytes with high concentration of beta-lactam antibiotics, cephaloridine (CER), flomoxef (FMOX) or cephamandole (CMD), resulted in significant reduction of cellular glutathione (GSH) levels, though cell viability was not affected during the incubation period. I.v. injection of a large dose, 300 mg/kg, of these beta-lactam antibiotics to rats did not affect the hepatic GSH levels. The concentration of beta-lactam antibiotics in the body fluid of the rats were found to be much lower than the amounts which causes GSH depletion in vitro.     

α-硫辛酸对高糖 高同型半胱氨酸诱导的小鼠足细胞凋亡的影响

目的探讨α-硫辛酸对高糖、高同型半胱氨酸(Hcy)作用下小鼠足细胞凋亡及Nephrin mRNA表达的影响。方法将体外培养的足细胞用高糖(25mmol/L)、Hcy(1mmol/L)、α-硫辛酸(0.5mmol/L)干预,分别在干预24、48、72h时收集细胞,流式细胞仪检测细胞凋亡,反转录聚合酶链反应(RT-PCR)法检测Nephrin mRNA表达。结果 24h时,高糖组、高Hcy组及混合组未见明显细胞凋亡(P>0.05);48h时,上述3组可见明显的细胞凋亡,且混合组凋亡更加明显(P<0.01);72h时,上述差异更加明显(P<0.01);α-硫辛酸可对抗高糖、高Hcy引起的足细胞凋亡(均P<0.05),且有一定的时间依赖性;高糖、高Hcy环境下,足细胞Nephrin mRNA表达呈下降趋势,且有一定的时效性;α-硫辛酸可对抗上述趋势。结论α-硫辛酸可减弱高糖、高Hcy对足细胞促凋亡作用,还可对抗其下调Nephrin mRNA的表达。     

Antioxidant and prooxidant activities of alpha-lipoic acid and dihydrolipoic acid

Reactive oxygen (ROS) and nitrogen oxide (RNOS) species are produced as by-products of oxidative metabolism. A major function for ROS and RNOS is immunological host defense. Recent evidence indicate that ROS and RNOS may also function as signaling molecules. However, high levels of ROS and RNOS have been considered to potentially damage cellular macromolecules and have been implicated in the pathogenesis and progression of various chronic diseases. alpha-Lipoic acid and dihydrolipoic acid exhibit direct free radical scavenging properties and as a redox couple, with a low redox potential of -0.32 V, is a strong reductant. Several studies provided evidence that alpha-lipoic acid supplementation decreases oxidative stress and restores reduced levels of other antioxidants in vivo. However, there is also evidence indicating that alpha-lipoic acid and dihydrolipoic acid may exert prooxidant properties in vitro. alpha-Lipoic acid and dihydrolipoic acid were shown to promote the mitochondrial permeability transition in permeabilized hepatocytes and isolated rat liver mitochondria. Dihydrolipoic acid also stimulated superoxide anion production in rat liver mitochondria and submitochondrial particles. alpha-Lipoic acid was recently shown to stimulate glucose uptake into 3T3-L1 adipocytes by increasing intracellular oxidant levels and/or facilitating insulin receptor autophosphorylation presumably by oxidation of critical thiol groups present in the insulin receptor beta-subunit. Whether alpha-lipoic acid and/or dihydrolipoic acid-induced oxidative protein modifications contribute to their versatile effects observed in vivo warrants further investigation.     

Enhancement by glutathione depletion of ethanol-induced acute hepatotoxicity in vitro and in vivo

Ethanol at initial concentrations between 0.75 and 6 g/l produced a dose-dependent release of the enzymes glutamic-pyruvic-transaminase and sorbitol dehydrogenase (GPT, SDH) from the isolated perfused rat liver. At the concentration of 6 g/l, it also decreased the oxygen consumption and elevated the calcium content of the isolated livers. These toxic effects of ethanol were significantly enhanced in livers, the glutathione content of which had been depleted by pretreatment with phorone. Ethanol-induced toxicity in glutathione-depleted isolated livers could be prevented both by inhibition of alcohol dehydrogenase with 4-methylpyrazole and of xanthine oxidase with allopurinol. In rats, in vivo, 1.6 g/kg ethanol injected intravenously produced a small increase in serum GPT and SDH concentrations 4 h after its administration. This increase in enzyme activities was several-fold higher and longer lasting in rats pretreated with phorone. Glutathione depletion per se did not induce hepatotoxicity in vitro or in vivo. Since glutathione is involved in several lines of defense against oxidative damage, our results of an enhanced susceptibility of glutathione-depleted livers to ethanol toxicity favour the hypothesis that ethanol exerts its hepatotoxic action via an activation of molecular oxygen.     

Effects of alpha-lipoic acid on deoxycorticosterone acetate-salt-induced hypertension in rats

We investigated the potential of natural occurring antioxidant alpha-lipoic acid to prevent hypertension and hypertensive tissue injury induced by deoxycorticosterone acetate (DOCA) and salt in rats. Two weeks after the start of DOCA-salt treatment, the rats were given alpha-lipoic acid (10 or 100 mg/kg/day, s.c.) or its vehicle for 2 weeks. Uninephrectomized rats without DOCA-salt treatment served as sham-operated controls. In vehicle-treated DOCA-salt rats, systolic blood pressure increased markedly after 3-4 weeks. Daily administration of 100 mg/kg alpha-lipoic acid for 2 weeks suppressed the increase in systolic blood pressure, whereas 10 mg/kg alpha-lipoic acid did not affect the progression of DOCA-salt-induced hypertension. When the degree of vascular hypertrophy of the aorta was morphometrically evaluated at 4 weeks, there were significant increases in media cross-sectional area in vehicle-treated DOCA-salt rats compared with sham-operated rats. The development of vascular hypertrophy was markedly suppressed by alpha-lipoic acid at 100 mg/kg but not at 10 mg/kg. Histopathological examination of the kidney in vehicle-treated DOCA-salt rats revealed fibrinoid-like necrosis in glomeruli and thickening of small arteries. In these animals, creatinine clearance decreased, and fractional excretion of Na(+), urinary excretion of protein and N-acetyl-beta-glucosaminidase increased. Such renal lesions and dysfunctions were ameliorated in DOCA-salt rats given alpha-lipoic acid. In addition, a marked increase in endothelin-1 content in both the aorta and kidney was evident in vehicle-treated DOCA-salt rats compared with findings in sham-operated rats. Significant attenuation of this increase occurred in alpha-lipoic acid-treated DOCA-salt rats. These results suggest that administration of alpha-lipoic acid to DOCA-salt hypertensive rats lessens the increased blood pressure and protects against renal and vascular injuries, possibly through the suppression of renal and vascular endothelin-1 overproduction.     

Protective effects of L-carnitine and alpha-lipoic acid in rats with adjuvant arthritis.

Free radicals play an important role in the pathophysiology of adjuvant arthritis. The purpose of this study was to assess the efficacy of L-carnitine (LC) and alpha-lipoic acid (alpha-LA) which are known to have antioxidant effects, in the treatment of adjuvant arthritis. Arthritis model was created by the administration of complete Freund's adjuvant (CFA) in 32 of 40 male Sprague-Dawley rats. The rats were divided into five groups. Rats in Group I served as controls and received 0.1 ml kg(-1) saline. Group II received only 0.1 ml of CFA and served as the CFA-control for the other groups. Groups III-V, after being injected with CFA, were treated with LC, alpha-LA or diclofenac, respectively. Levels of malondialdehyde (MDA) and glutathione (GSH) were measured in plasma samples. Enzyme activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) were measured. The paws of rats were evaluated histopathologically to investigate the anti-inflammatory effects. TNF-alpha levels were measured for the evaluation of inflammation. In Group II plasma MDA increased, levels of glutathione decreased, enzyme activities of SOD and GPx decreased. Histopathological damage increased in the paws of the rats in this group. MDA levels decreased in Groups III-V when compared with Group II. GSH levels significantly increased in Group III and IV than Group V. SOD activity of Group IV was higher than Group III and V. TNF-alpha levels were significantly lower in Group IV and V. LC and alpha-LA seemed to have protective effects against oxidative damage in adjuvant arthritis model.     

Nicotinic acid and pyridoxine modulate arachidonic acid metabolism in vitro and ex vivo in man.

The in vitro effects of nicotinic acid (10-1000 microM), pyridoxine (0.1-500 microM) and pyridoxal-5'-phosphate (0.1-500 microM) and the ex vivo effects of nicotinic acid (2500 mg orally during 12 h) and pyridoxine (600 mg orally daily for seven days) on arachidonic acid metabolism were investigated in calcium ionophore A23187 (calcimycin)-stimulated human whole blood. In vitro nicotinic acid stimulated prostaglandin E2, thromboxane B2 and leukotriene E4 synthesis. Pyridoxine at all concentrations and pyridoxal-5'-phosphate at the highest concentration stimulated prostaglandin E2 and thromboxane B2 production, but had no effect on leukotriene E4 synthesis. Nicotinic acid treatment increased ex vivo prostaglandin E2, thromboxane B2 and leukotriene E4 synthesis to 185%, 165% and 175% of the initial values, respectively. In the pyridoxine-treated subjects, ex vivo prostaglandin E2, thromboxane B2 and leukotriene E4 synthesis was decreased after seven days to 75%, 65% and 45% of the initial values, respectively. In the present study the effects of nicotinic acid on the 5-lipoxygenase pathway in arachidonic acid metabolism were studied for the first time and the drug was found to stimulate this pathway in vitro and ex vivo. In vitro pyridoxine and pyridoxal-5'-phosphate had no effect on the 5-lipoxygenase pathway. The inhibition of leukotriene synthesis by pyridoxine ex vivo might be of therapeutic importance.     

Quinolinic acid effects on amino acid release from the rat cerebral cortex in vitro and in vivo.

1. The effect of quinolinic acid, N-methyl-D,L-aspartate (NMDLA) and kainate on the release of endogenous and exogenous amino acids from the rat cerebral cortex in vitro and in vivo was studied. 2. Neither quinolinic acid nor NMDLA had any effect on the basal or potassium-evoked release of [3H]-D-aspartate from slices of rat cerebral cortex either in the presence or absence of magnesium. Kainic acid failed to modify the basal efflux of [3H]-D-aspartate but significantly inhibited (by 34.4% +/- 0.04%, P less than 0.05) the potassium-evoked release. 3. Neither quinolinate nor NMDLA had any effect on the basal efflux of endogenous amino acids from rat cortical slices either in the presence or absence of magnesium ions at concentrations between 10 microM and 5 mM. 4. Both NMDLA (1 mM) and quinolinate (5 mM) produced an efflux of endogenous aspartate (371.4% +/- 11.6%; 389.3% +/- 12.1%) and glutamate (405.4% +/- 13.6%; 430.1 +/- 8.7%) respectively from the rat cerebral cortex in vivo (P less than 0.01). The quinolinic acid-evoked efflux was abolished by the NMDLA antagonist, 2-amino-5-phosphonovaleric acid (200 microM). 5. Kainic acid also caused an efflux of endogenous amino acids from the rat cerebral cortex in vivo. However, the profile of this release was different from that produced by quinolinate and NMDLA. 6. The results add further support to the suggestion that quinolinic acid acts at the NMDLA-preferring receptor and may also explain the requirement for intact afferent projections for the neurotoxic effects of quinolinate to be manifested.     

In vivo and in vitro hepatotoxicity and glutathione interactions of N-methyldithiocarbamate and N,N-dimethyldithiocarbamate in the rat.

The ability of N-methyldithiocarbamate (NMDC) to generate methylisothiocyanate and HS(-) together with its greater acid stability suggest that NMDC may exert greater acute toxicity following oral exposure than its dialkyl analog,N,N-dimethyldithiocarbamate (DMDC). To assess this possibility, cell culture, perfused liver, and in vivo studies were performed to delineate differences in the hepatotoxicity and thiol interactions of NMDC and DMDC in the rat. The role of methylisothiocyanate and HS(-) in NMDC-induced hepatotoxicity was evaluated and glutathione interactions characterized through analysis of reduced glutathione (GSH), glutathione disulfide (GSSG), and S-methylthiocarbamoylglutathione (GSMITC) using HPLC and liquid chromatography tandem mass spectrometry (LC/MS/MS). Following oral administration, centrilobular hepatocyte necrosis and enzyme leakage was observed for NMDC but not for DMDC. Dose dependent decreases of intracellular GSH were produced by both dithiocarbamates in primary hepatocytes but DMDC appeared to deplete GSH through the generation of GSSG whereas NMDC produced GSMITC consistent with the generation of a methylisothiocyanate intermediate. In primary hepatocytes, both NMDC and DMDC cytotoxicity was increased by prior depletion of intracellular GSH and diminished by prior supplementation of GSH. The results obtained using perfused livers were similar for NMDC in that elevated levels of GSMITC were detected in the bile; however, DMDC produced only a modest increase of GSSG over controls that was not significantly different to that produced by NMDC. Results obtained from isolated liver mitochondria and primary hepatocytes were not consistent with NMDC producing HS(-)-mediated inhibition of mitochondrial respiration. These data support a greater potential for hepatotoxicity to result following oral exposure to NMDC relative to DMDC and that glutathione may play a role in cytoprotection for NMDC, presumably through detoxification of a methylisothiocyanate metabolite.     

Effects of verapamil on gastric acid secretion in vitro and in vivo

The activity of H,K-ATPase, the gastric acid producing enzyme, was concentration dependently inhibited by verapamil in the mM range. Verapamil concentration dependently inhibited acid formation in gastric glands, measured as [14C]aminopyrine accumulation or oxygen consumption. The IC50 values were in the microM range. No inhibition of acid secretion by verapamil was observed in Heidenhain-pouch dogs and stomach-lumen-perfused rats. However, in pylorus-ligated rats an inhibition was observed, this effect is related to its cardiovascular effectiveness. To understand the action of verapamil, its physicochemical properties were considered. Verapamil is a highly lipophilic base with a pKa of 8.7. It accumulates in membranes and in the acidic spaces of the parietal cell. We suggest that the inhibition of vesicular bound H,K-ATPase is dependent on a non-specific accumulation of verapamil in the membrane (detergent effect) and that inhibition of acid production in vitro is due to an additional accumulation of the drug in acidic compartments, leading to an impaired function of the proton pump. Verapamil does not decrease acid secretion in vivo by this mechanism as the required dose would be higher than the dose that causes a strong depression of the cardiovascular system.     

Structural characterization of in vivo rat glutathione adducts and a hydroxylated metabolite of simvastatin hydroxy acid.

Simvastatin hydroxy acid (SVA), the pharmacologically active form of simvastatin (SV), is a potent inhibitor of 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A reductase and is formed on hydrolysis of the orally administered SV. In this article, we report the structural characterization of two new dihydroxy glutathione adducts and a trihydroxy derivative of SVA, all found in rat bile. Metabolite I is 5'beta,6'beta-dihydroxy-4'a(alpha)-glutathione-SVA, and metabolite II is a pentanoic acid derivative of metabolite I. The two identified GSH conjugates accounted for 16 and 9% in males and 11 and 5% in females of the total radioactivity (metabolites I and II, respectively). Metabolite III is 3',5'beta,6'beta-dihydrotriol-SVA and accounts for 2% (male) and 4% (female) of the total dose in rats. Of these three newly identified metabolites, only metabolite III was also observed in dog bile.     

Conjugation of butadiene diepoxide with glutathione yields DNA adducts in vitro and in vivo

1,2,3,4-Diepoxybutane (DEB) is reported to be the most potent mutagenic metabolite of 1,3-butadiene, an important industrial chemical and environmental pollutant. DEB is capable of inducing the formation of monoalkylated DNA adducts and DNA-DNA and DNA-protein cross-links. We previously reported that DEB forms a conjugate with glutathione (GSH) and that the conjugate is considerably more mutagenic than several other butadiene-derived epoxides, including DEB, in the base pair tester strain Salmonella typhimurium TA1535 [Cho et al. (2010) Chem. Res. Toxicol. 23, 1544-1546]. In the present study, we determined steady-state kinetic parameters of the conjugation of the three DEB stereoisomers-R,R, S,S, and meso (all formed by butadiene oxidation)-with GSH by six GSH transferases. Only small differences (<3-fold) were found in the catalytic efficiency of conjugate formation (k(cat)/K(m)) with all three DEB stereoisomers and the six GSH transferases. The three stereochemical DEB-GSH conjugates had similar mutagenicity. Six DNA adducts (N(3)-adenyl, N(6)-adenyl, N(7)-guanyl, N(1)-guanyl, N(4)-cytidyl, and N(3)-thymidyl) were identified in the reactions of DEB-GSH conjugate with nucleosides and calf thymus DNA using LC-MS and UV and NMR spectroscopy. N(6)-Adenyl and N(7)-guanyl GSH adducts were identified and quantitated in vivo in the livers of mice and rats treated with DEB ip. These results indicate that such DNA adducts are formed from the DEB-GSH conjugate, are mutagenic regardless of sterochemistry, and are therefore expected to contribute to the carcinogenicity of DEB.     

Relationship between the intracellular accumulation of anthracyclines and effectiveness in vitro and in vivo

The relationship between accumulation, retention and cytotoxicity of various anthracyclines was investigated in Friend leukaemia cells growing in vitro. By comparison to that of adriamycin (ADM) and epi-adriamycin (Epi-ADM), the uptake of demethoxy-daunorubicin (DM-DNR) and THP-adriamycin (THP-ADM) is a rapid process. In cells exposed to DM-DNR or THP-ADM, a 50% accumulation is reached in less than 2 min, whereas 80 min and up to 4 hours are needed for epi-ADM or ADM, respectively. More than 95% of these anthracyclines are accumulated and retained in the nuclear fraction. Following a short cell exposure, the intracellular concentrations of the rapidly incorporated drugs (DM-DNR or THP-ADM) decrease with the cell density. After cell exposure to one of these drugs followed by growth in drug-free medium, the cytotoxic activity is related to the ease with which the anthracycline accumulates in cells. Since the pharmacological properties of these anthracyclines differ, and because cytotoxic activity correlates with these properties, plasma and intracellular concentrations of ADM and THP-ADM were studied after intravenous administration in leukaemic and non-leukaemic patients with various white blood cell concentrations. Since the pharmacokinetic studies in vivo correlated to in vitro parameters, it is concluded that administration modalities have to be determined, adapted to each patient, and considered differently according to the anthracycline used.