Effects Of Diethyl Maleate Research Articles

Effect of diethyl maleate on the toxicity of essential oil from Citrus reticulata fruit peels to Tribolium castaneum Herbst under laboratory conditions

Certain compounds of plants, essential oils, with insecticidal properties have been considered as alternatives to chemical pesticides for pest control in recent years. In this study, the synergistic effect of diethyl maleate (DEM) on the toxicity of Citrus reticulata Blanco (Rutaceae) peel essential oil against a stored-product insect pest, i.e. red flour beetle, Tribolium castaneum Herbst (Tenebrionidae) adults was studied. DEM [one part], combined with acetone [two parts], was applied on T. castaneum adults. Five concentrations of essential oil from C. reticulata were tested. Three replicates and 30 adult insects/replicate/each concentration were used. LC50 values after 24 and 48 h of exposure were 33.8 and 28.2 μl/l air, respectively. Combination of the essential oil from C. reticulata with the synergist DEM after 24 and 48 h of exposure decreased the corresponding LC50 values to 18.1 and 12.2 μl/l air, respectively. These results revealed that DEM can considerably improve the potency of essential oil from C. reticulata and maybe applied successfully in the stored-product pest control programmes.

Disulfiram-like effect of diethyl maleate on barbiturate-induced hypnosis and 5-hydroxytryptamine metabolism.

Diethyl maleate (DEM, 600 mg kg-1 i.p.) significantly potentiated hexobarbitone hypnosis and lowered plasma hexobarbitone level on awakening. Sleeping time following intracerebroventricular (i.c.v.) injection of phenobarbitone was also prolonged by DEM treatment. When administered to DEM-treated rats, L-tryptophan (50 mg kg-1 i.p.) significantly potentiated hexobarbitone hypnosis, although alone it had no effect in control rats. DEM markedly diminished the activity of brain low-Km aldehyde dehydrogenase (A1DH) and the formation of 5-hydroxyindoleacetic acid from 5-hydroxytryptamine (5-HT) without affecting MAO activity in various areas of the brain. Conversely, the protein-bound radioactivity derived from i.c.v. [14C]-5-HT was increased by DEM treatment. These results showed that DEM is comparable with disulfiram, a brain A1DH inhibitor, in terms of its effect on 5-HT metabolism and barbiturate hypnosis.

Real‐time measurement of cytosolic free calcium concentration in DEM‐treated HL‐60 cells during static magnetic field exposure and activation by ATP

This study investigated whether glutathione depletion affected the sensitivity of HL-60 cells to static magnetic fields. The effect of Diethylmaleate (DEM) on static magnetic field induced changes in cytosolic free calcium concentration ([Ca(2+)](c)) was examined. Cells were loaded with a fluorescent dye and exposed to a uniform static magnetic field at a strength of 0 mT (sham) or 100 mT. [Ca(2+)](c) was monitored during field and sham exposure using a ratiometric fluorescence spectroscopy system. Cells were activated by the addition of ATP. Metrics extracted from the [Ca(2+)](c) time series included: average [Ca(2+)](c) during the Pre-Field and Field Conditions, peak [Ca(2+)](c) following ATP activation and the full width at half maximum (FWHM) of the peak ATP response. Comparison of each calcium metric between the sham and 100 mT experiments revealed the following results: average [Ca(2+)](c) measured during the Field condition was 53 +/- 2 nM and 58 +/- 2 nM for sham and 100 mT groups, respectively. Average FWHM was 51 +/- 3 s and 54 +/- 3 s for sham and 100 mT groups, respectively. An effect of experimental order on the peak [Ca(2+)](c) response to ATP in sham/sham experiments complicated the statistical analysis and did not allow pooling of the first and second order experiments. No statistically significant difference between the sham and 100 mT groups was observed for any of the calcium metrics. These data suggested that manipulation of free radical buffering capacity in HL-60 cells did not affect the sensitivity of the cells to a 100 mT static magnetic field.

The dietary antioxidant resveratrol affects redox changes of PPARα activity

Background and aims Gene–environment interaction is behind the pathogenesis of most widespread diseases, and nutrition is among the environmental factors with the highest impact on human health. The mechanisms involved in the interaction between nutritional factors and the genetic background of individuals are still unclear. The aim of this study was to investigate whether resveratrol (RES), an antioxidant polyphenol of red wine, can influence the activity of PPARα in the rat hepatoma cell line McArdle-RH7777. PPARα is a transcriptional factor that regulates gene expression when activated by endogenous or exogenous long-chain fatty acids. Its activation results in significant protection from cardiovascular diseases in humans. Methods and results By means of the electromobility shift assay (EMSA), we observed that PPARα is redox-sensitive as it displays reduced DNA-binding activity following in vivo treatment of the cells with 1 mmol/L diethylmaleate (DEM), a glutathione-depleting agent. This finding could be relevant considering the important role of redox balance in pathological and physiological processes. We also observed a dual effect of 100 μmol/L RES on PPARα activity: it was able to prevent, to a large extent, the DEM-induced reduction of DNA-binding activity at earlier time points, when the effect of DEM was stronger, but it depressed PPARα activity at later time points, when the effect of DEM was greatly reduced. Conclusion A nutritional substance, such as RES, is able to influence the activity of gene-regulating factors, but the net effect is difficult to predict when the compound involved has multiple biological properties. Caution is therefore warranted before drawing conclusions about the potential benefits of RES for human health.

Protein Kinase B Activation by Reactive Oxygen Species Is Independent of Tyrosine Kinase Receptor Phosphorylation and Requires Src Activity

Reactive oxygen species (ROS) participate as second messengers in the mitogenic signal transduction. Most of the experimental data supporting the role of ROS as signaling molecules have been obtained by using H2O2. Exposure of cells to H2O2 rapidly increases tyrosine phosphorylation of tyrosine kinase receptors (TKRs) in the absence of growth factor binding, thus inducing the activation of downstream signaling cascades, like that of protein kinase B (AKT). Another molecule able to induce an increase of intracellular ROS levels is diethylmaleate (DEM), which acts by depleting the ROS scavenger reduced glutathione (GSH). A comparison of the effects exerted by H2O2 and DEM shows that the latter induces redox modifications milder than those generated by H2O2. We also demonstrated that DEM-induced redox modifications are not accompanied by platelet-derived growth factor-receptor (PDGF-R) and epidermal growth factor-receptor Tyr phosphorylation, although they are able to activate ERKs and AKT, with kinetics different from those observed following H2O2 treatment. The activation of these two pathways is not blocked by AG1296, a selective inhibitor of PDGF-R Tyr kinase, thus confirming that the effects of DEM are not mediated by the TKR phosphorylation. On the contrary, PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazole[3,4-d]pyrimidine), an inhibitor of Src kinase, completely prevents DEM- and H2O2-induced AKT activation but has no effect on the pathway of ERKs. Finally, nitration of Tyr residues in PDGF-R is observed in DEM-treated cells, thus suggesting that ROS-induced modifications different from Tyr phosphorylation can occur at the growth factor-receptor level and can be involved in the regulation of signaling pathways.

Effects of diethylmaleate on DNA damage and repair in the mouse brain

The enzyme 8-oxoguanine DNA glycosylase 1 participates in the repair of damaged DNA by excising the oxidized base 8-hydroxy-2′-deoxyguanosine. We have previously demonstrated that enzymatic activity of this enzyme is inversely related to the levels of the damaged base in specific brain regions. We now report that the activity of 8-oxoguanine DNA glycosylase 1 is increased in a region-specific manner following treatment with diethylmaleate, a compound that reduces glutathione levels in the cell. A single treatment with diethylmaleate elicited a significant increase (∼2-fold) in the activity of 8-oxoguanine DNA glycosylase 1 in three brain regions with low basal levels of activity (cerebellum, cortex, and pons/medulla). There was no change in the activity of 8-oxoguanine DNA glycosylase 1 in those regions with high basal levels of activity (hippocampus, caudate/putamen, and midbrain). This is the first report to demonstrate that DNA repair capacity can be upregulated in the CNS, and the increased repair activity correlates with a reduction in the levels of DNA damage. The brain region-specific capacity to deal with increased oxidative damage to DNA may be responsible, in part, for the vulnerability of specific neuronal populations with aging, sources of oxidative stress, and neurodegenerative diseases.

Composites from functionalized polyolefins and silica

The effect of diethyl maleate (DEM) units grafted to the polyolefins on the compatibility with inorganic fillers has been investigated. Ethylene-propylene rubber functionalized with DEM (EPR-g-DEM) was mixed with silica in different weight ratios using a Brabender mixer (Plastograph), by comparing analogous mixtures prepared starting with unmodified ethylene-propylene-rubber (EPR) and silica. The Torque traces showed that the diethyl maleate grafted on the EPR significantly changed the interfacial interaction between the silica surface and the EPR during the mixing process. Incorporation of silica into either EPR and EPR-g-DEM by mechanical mixing produced a fraction of insoluble by association with the filler (bound rubber), as revealed by the solvent extraction technique. Bound rubber evaluations pointed out the major capability of the EPR-g-DEM to interact with the mineral surface. In this case specific interactions involving the carbonyl groups of the functionalized polyolefin and the polar sites on the filler surface have been identified by means of FT-IR spectroscopy. As revealed by SEM observations, these specific interactions at the interface promote the mutual wettability and adhesion of the components in EPR-g-DEM/silica composites.

Priming prostate carcinoma cells for increased apoptosis is associated with up-regulation of the caspases.

The potential to prime prostatic carcinoma cell lines for apoptosis represents an exciting strategy for the treatment of patients with this disease. The ability and the underlying molecular mechanisms involved in sensitizing both androgen-sensitive and androgen-insensitive cell types to a range of apoptotic-inducing agents are investigated by the authors. Primary and secondary cell lines were pretreated with diethyl-maleate (DEM) prior to the induction of apoptosis by Fas antibody (1 microg/mL), cycloheximide (1 microg/mL), etoposide (62.5 microM), and radiation (5 grays). It was demonstrated previously that DEM (50 microM) increases the sensitivity to apoptosis induced by these agents. The effects of DEM on both protein and RNA expression was determined by Western blot analysis and a ribonuclease protection assay, respectively. The effects of DEM on intracellular glutathione (GSH) levels and its intracellular distribution also were assessed. DEM did not affect the expression of the caspases at the transcriptional level but was associated with increased procaspase-3 and caspase-8 protein levels. DEM preincubation restored sensitivity to Fas antibody and radiation-induced apoptosis in cells from the LNCaP-bcl-2 transfectant cell line that, normally, are resistant to these apoptotic stimuli. It is that DEM chemically depletes intracellular thiol levels. Although no depletion in total intracellular thiol GSH was observed at these concentrations of DEM, trafficking of GSH from the nucleus to the cytosol was demonstrated. Identification of the caspases as a potential target for chemical manipulation may serve as an effective, adjuvant-based approach in the treatment of patients with prostate carcinoma and, in particular, for immunotherapy and radiation-based strategies that rely on the activation of these death-effector proteases.

GLUTATHIONE DEPLETION-INDUCED NEUTROPHIL APOPTOSIS IS CASPASE 3 DEPENDENT

Resolving inflammation is a vital step in preventing the persistence of inflammatory disorders. Neutrophils play a major role in tissue damage associated with an inflammatory response. Their death by apoptosis is central to the final resolution of this response. Thiol depletion with diethylmaleate (DEM) or diamide represent important triggers for neutrophil apoptosis. The mechanism by which this process occurs remains unknown. The apoptotic cascade is associated with a number of cellular changes, including caspase activation and mitochondrial permeability. The aims of this study were to determine the role of mitochondrial permeability and the caspase cascade in thiol depletion-induced neutrophil apoptosis. Total cellular glutathione was reduced by DEM and diamide. This reduction was associated with neutrophil apoptosis and an increase in caspase 3 activity. The effects of DEM were blocked by the caspase 3 inhibitor, Z-DEVD-FMK. Mitochondrial permeability that occurred was also increased during this induction of apoptosis. Bongkrekic acid, a mitochondrial membrane stabilizer, inhibited DEM-induced apoptosis. The inhibitors' effects of LPS or GM-CSF on spontaneous neutrophil apoptosis was reversed by DEM, which was mediated by an increase in caspase 3 activity and independent of mitochondrial disruption. Caspase activation is an important step in glutathione depletion-induced apoptosis in resting and inflammatory neutrophils. Regulation of caspase activity may represent a possible target to trigger apoptosis and resolve inflammatory disorders.

Effect of glutathione deficiency on the adipocyte sn-glycerol-3-phosphate acyltransferase

The present study investigates the effects of various glutathione (GSH) depleting agents on sn-glycerol-3-phosphate acyltransferase (GPAT) activity, the first committed step in adipose triacylglycerol formation. GPAT activity was measured in the presence of [ 14C]glycerol-3-phosphate and palmitoyl-CoA, using different subcellular fractions. Glutathione deficiency in animals was induced in the presence of diethylmaleate (DEM) or buthionine sulfoximine. In this respect, DEM (1.75 mmoles/kg) was more effective and caused over 75% decrease in GPAT activity within 4 h of DEM administration. Further studies indicated that this decrease in GPAT activity was mainly related to the microsomal form of GPAT, without any significant effect on mitochondrial GPAT activity. Adipocytes incubated with 2.5 mm DEM for 1 h at 37°C also showed a reduction in the adipocyte glutathione content, which was accompanied by decreases in GPAT activity. The effect of DEM on adipocyte GPAT activity was partially reversible in the presence of cell permeable glutathione ethyl ester. Preincubation of adipose tissue homogenates with 2.5 mM DEM at 30°C for 45 min also showed a significant loss of the GPAT activity. The presence of 5 mM dithiothreitol in the preincubation mixture offered a significant protection of the GPAT activity against DEM. However, glutathione was ineffective in this respect as it interfered with the utilization of palmitoyl-CoA in the GPAT assay. Therefore, on the basis of these three different approaches, the present studies suggest that the thiol environment offered by glutathione (in vivo and in vitro studies) or dithiothreitol (in a cell-free system) is critical for the maintenance of GPAT activity.

Enhancement of expression of stress proteins by agents that lower the levels of glutathione in cells

The effects of diethyl maleate and buthionine sulfoximine, agents that lower cellular levels of glutathione, on expression of hsp27 and αB crystallin in response to stress were studied. When C6 rat glioma cells were treated with 100 μM arsenite for 1 h, accumulation of the two proteins, estimated by specific immunoassays, was markedly enhanced by additional exposure to 1 mM diethyl maleate or 2.5 mM buthionine sulfoximine. The latter also increased heat- and CdCl 2-induced accumulation of hsp27 and αB crystallin. Stress-induced accumulation of hsp70, estimated by Western blotting analysis, was also enhanced by these agents. Northern blotting analysis revealed increase in levels of mRNAs for hsp27, αB crystallin and hsp70. The period of heat shock element (HSE)-binding activity of heat shock factor (HSF) stimulated by arsenite was extended by addition of diethyl maleate and buthionine sulfoximine. The induced phosphorylated state of HSF1 was also prolonged by diethyl maleate. Although exposure of cells to diethyl maleate alone for 1 h caused neither accumulation of hsp27, αB crystallin and hsp70 nor expression of mRNAs for these proteins, HSE-binding activity of HSF was stimulated. However, the activated HSF was not phosphorylated. These results suggest that diethyl maleate induces an intermediate state of HSF that binds to HSE but is transcriptionally inert. The mechanism is unclear but the levels of glutathione in cells that were exposed to diethyl maleate or buthionine sulfoximine were markedly decreased.

Effect of diethylmaleate on liver extracellular glutathione levels before and after global liver ischemia in anesthetized rats

Glutathione (GSH), present in a high concentration in the liver, serves important protective functions. We investigated the effect of lowered tissue GSH content, accomplished by diethylmaleate (DEM) administration, on liver extracellular GSH levels before and after global ischemia in anesthetized rats. Liver extracellular GSH levels were determined by microdialysis perfusion and an on-line high performance liquid chromatography system. Global liver ischemia was induced by ligation of the hepatic pedicles including the hepatic artery, portal vein, and bile duct. DEM (4 mmol/kg) significantly lowered both the liver tissue GSH levels (1.36 ± 0.26 μmol/g wet wt vs 9.50 ± 0.55 μmol/g wet wt for the untreated) and the liver extracellular GSH levels (4.3 ± 2.4 μM vs 25.2 ± 8.7 μM for the untreated). Global liver ischemia induced a dramatic increase in the liver extracellular GSH level. Although the liver tissue GSH level was lowered following DEM treatment, DEM administration did not affect significantly ischemia-induced elevation of extracellular GSH (when presented as fold increase relative to basal value). In conclusion, DEM showed a direct effect on liver extracellular GSH content in anesthetized rats. However, DEM treatment did not affect the relative release of GSH following global liver ischemia.

Detection of Free Radical Metabolite Formation Usingin VivoEPR Spectroscopy: Evidence of Rat Hemoglobin Thiyl Radical Formation Following Administration of Phenylhydrazine

The spin-trapping technique in conjunction with a low-frequency electron paramagnetic (or spin) resonance (EPR or ESR) spectrometer was used to detect the hemoglobin thiyl free radical in living rats using a whole body resonator. The hemoglobin thiyl free radical was formed following the intragastric administration of phenylhydrazine at the LD50dose of 188 mg/kg. The hemoglobin thiyl free radical was then trapped by preinjected 5,5-dimethyl-1-pyrrolineN-oxide (DMPO), which formed the DMPO/hemoglobin thiyl-free radical adduct in the blood. The time course of thein vivoformation and disappearance of the spin adduct was followed. The DMPO/hemoglobin thiyl free radical was detected in blood samples using 9.5 GHz (X-band) and 1.1 GHz (L-band) EPR at room temperature and 77 K. Pretreatment of rats with ascorbate and diethylmaleate (DEM) decreased the signal intensity of the DMPO/hemoglobin thiyl free radical spin adduct. The incubation of ascorbate or DEM at 37°C with rat blood containing preformed DMPO/hemoglobin thiyl radical adduct showed that there was no effect of DEM on the free radical concentration, while ascorbate reduced the radical adduct. This study provided direct evidence of the formation of the DMPO/hemoglobin thiyl free radicalin vivoand enabled us to study this formation in living animals free of any artifacts that can occur when usingex vivomethods.

Diethyl maleate, an in vivo chemical depletor of glutathione, affects the response of male and female rats to arsenic deprivation

An experiment was performed to determine the effect of diethyl maleate (DEM), an in vivo depletor of glutathione, on the response of male and female rats to arsenic deprivation. A 2 x 2 x 2 factorially arranged experiment used groups of six weanling Sprague-Dawley rats. Dietary variables were arsenic at 0 or 0.5 microgram/g and DEM at 0 or 0.25%; the third variable was gender. Animals were fed for 10 wk a casein-ground corn based diet that contained amounts of calcium, phosphorus, and magnesium similar to the AIN-76 diet. DEM supplementation increased blood arsenic in both male and female rats; female rats had the greatest amount of arsenic in whole blood. Although female rats in general had a lower concentration of glutathione in liver, those fed no supplemental DEM, regardless of their arsenic status, had the lowest amounts. Compared to males, female rats had a lower activity of liver glutathione S-transferase (GST). Arsenic deprivation decreased, and DEM supplementation increased liver GST activity in both male and female rats. Lung GST activity was also increased by DEM supplementation in male, but not female, rats. The most striking finding of the study was that compared to males, females had extremely elevated kidney calcium concentrations, and that the elevation was exacerbated by arsenic deprivation. DEM supplementation also exacerbated the accumulation of calcium in the kidney of the female rats. The response of the rat to both DEM and arsenic was, for many variables, dependent on gender. This gender dependence may be explained by the differences in methionine metabolism between male and female rats. Thus, arsenic deprivation apparently can manifest itself differently depending on gender.

Effect of diethyl maleate on glutathione, hepatic and renal cortical perfusion, and portal 6-ketoPGF 1α and TxB 2 levels in swine

1. Effects of diethyl maleate (DEM) mediated glutathione (GSH) depletion on hepatic and renal cortical blood flow (perfusion), plasma GSH, and portal prostacyclin (6-ketoPGF 1α) and thromboxane (TxB 2) were determined in anaesthetized swine. 2. Although DEM depleted hepatic GSH to 25% of control, plasma GSH increased 10-fold in comparison to controls. DEM caused a drop in blood pressure and renal cortical perfusion but had no effect on hepatic perfusion or portal 6-ketoPGF 1α or TxB 2 levels. 3. Possibly, the unexpected rise in plasma GSH may have inhibited prostanoid synthesis, preventing any alterations in tissue perfusion that may have occurred following tissue GSH depletion.

Protein damage and lipid peroxidation: Effects of diethyl maleate, bromotrichloromethane and vitamin E on ammonia, urea and enzymes involved in ammonia metabolism

Changes in ammonia and urea were investigated as potential marker products of free radical damage to protein and subsequent metabolism of those damaged proteins in vivo. Both serum and liver lipid peroxidation products as measured by thiobarbituric-acid-reactive substances (TBARS) were increased by feeding rats a vitamin-E-deficient diet. The acute injection of diethyl maleate and bromotrichloromethane (DEM/ BrCCl 3) increased TBARS in liver of rats fed a vitamin-E-deficient diet. The concentrations of ammonia and urea in the serum and liver did not correlate with lipid peroxidation. The activities of liver glutaminase and arginase were decreased by DEM/BrCCl 3 treatment in rats fed vitamin-E-deficient diet. Glutamateammonia ligase activity was decreased by vitamin-E-deficient diet but not by DEM/BrCCl 3 treatment. Omithine carbamoyltransferase, arginosuccinate synthase, argininosuccinate lyase and glutamate dehydrogenase (NAD(P) +) were not affected by dietary vitamin E or by DEM/BrCCl 3. The data suggest that the concentrations of ammonia and urea, major by-products of nitrogen metabolism, are unchanged by the oxidant damage and lipid peroxidation, and that their control in vivo is a dynamic equilibrium of various metabolic pathways.

Effects of diethyl maleate (DEM), a glutathione depletor, on prostaglandin synthesis in the isolated perfused spleen of rabbits

To investigate the role of glutathione (GSH) on prostaglandin (PG) synthesis, isolated rabbit spleens were perfused with Tyrode's solution with or without the addition of diethyl maleate (DEM) in concentrations up to 1 mM. In the absence of DEM, PG synthesis was stimulated by the Ca2+ ionophore A23187 (20 nmole) or arachidonate (0.4 mumole). Prostaglandin (PG) E2 was a major product, accounting for 60-70% of the total cyclooxygenase products. Small amounts of PGF2 alpha, 6-keto-PGF1 alpha, PGD2 and thromboxane (Tx) B2 were also produced. When DEM was added to the perfusion medium, GSH content decreased dose-dependently with increasing DEM concentration. Lactate dehydrogenase activity was not detected in the venous effluent, indicating that DEM depleted intrasplenic GSH without causing any lysis of cellular membranes. A23187-induced production of PGs and of Tx was decreased with increasing concentrations of DEM up to 0.5 mM, whereas at 1.0 mM DEM, these products showed a tendency to increase as compared with levels at 0.5 mM DEM. However, this increase was only significant for TxB2, which returned to levels obtained in the absence of DEM. DEM 1 mM did not cause cell lysis, but it appears to perturb the cell membrane to a degree similar to that which occurs with stimulation of phospholipase A2. The small but significant increase of TxB2 with 1.0 mM DEM could be a result of decreased PGE2 isomerase activity. Perfusion with arachidonate gave virtually identical results: 1.0 mM DEM attenuated the production of all prostanoids except for TxB2 as compared with untreated controls. These results suggest that GSH contributes to the regulation and/or maintenance of PGs synthesis.

Effect of phenobarbital, naphthoflavone and diethlmaleate on the sex dependent urinary pattern of hexachlorobenzene metabolites in the rat

The sex related difference in the urinary excretion of pentachlorothiophenol (PCThP) was investigated in rats after a single oral dose of hexachlorobenzene (HCB) and compared to that obtained after a single oral dose of pentachloronitrobenzene (PCNB). A ten fold difference was obtained in both cases in favour of females. The effect of phenobarbital (PB) and β-naphtoflavone (β–NF) and the effect of diethylmaleate (DM) on the urinary pattern of HCB metabolites was investigated in male and female rats. PB enhanced the urinary excretion of pentachlorophenol (PCP) and tetrachlorohydroquinone (TCHQ) but had no effect on the excretion of PCThP. β-NF did not have any effect on any of the three main metabolites while an hepatic glutathione depletion induced by DM significantly reduced the excretion of PCThP in females. The results suggest that, after HCB or PCNB intake, a common sex dependent biotransformation step leads to the formation of PCThP, after conjugation with glutathione, independently of the pathway leading to the formation of PCP which is under the control of a PB inducible form of cytochrome P-450.

Effect of diethyl maleate and sodium salicylate on cefmetazole transport in the in vitro rat everted intestinal sac: influence of Ca2+ and vanadate in the media.

InCa2+-containing media, 1 mm diethyl maleate (DEM) increased the in vitro cefmetazole transport in rat everted colonic sac rapidly after a significant nonprotein thiol loss, but the increase in the ileal sac occurred only slowly after a significant nonprotein thiol loss. However, Ca2+-free media promoted the adjuvant effect of DEM rapidly even in the ileal sac. Since vanadate in the Ca2+-containing media also promoted the adjuvant effect of DEM, it is suggested that a perturbation of Ca2+ homeostasis as a secondary effect after nonprotein thiol loss is related to the increase of the intestinal mucosal permeability and that the ileal mucosa has stronger resistance to the secondary effect than the colonic mucosa does. The adjuvant effect of 300 mm salicylate (SA) was also promoted in Ca2+-free media. Since the effect of SA was stronger than that of DEM even though SA caused less nonprotein thiol loss, it is considered that the effect of SA involves at least two mechanisms.

Changes in biliary secretion and lactate metabolism induced by diethyl maleate in rabbits

Diethyl maleate is a compound which binds with glutathione by means of a glutathione S-transferase and is excreted into bile leading to a rapid depletion of hepatic glutathione. In the rabbit, the activity of the enzyme is fairly low and we were thus prompted to study the possible effects of diethyl maleate on biliary secretion and metabolic status in this species. The administration of diethyl maleate induced a transient choleresis followed by cholestasis. The choleresis coursed with increases in the biliary output of sodium and unacounted anions, whereas those of chloride, bicarbonate and bile acids were unaffected. Our data seem to confirm that choleresis is due to the osmotic activity of diethyl maleate compounds excreted into bile, as has been reported in rats and dogs. The cholestasis observed coursed with falls in the outputs of sodium, chloride and bicarbonate though that of bile acids remained constant. Following diethyl maleate administration, a metabolic acidosis appeared with progressive increases of blood lactate concentration. In bile the concentration of this anion closely followed that of plasma. The cholestasis is attributed to a lowered biliary secretion of bicarbonate probably secondary to the metabolic alteration. The hepatic values of cytoplasmic and mitochondrial NADH/NAD ratios and of adenine nucleotide concentrations suggest that the increase in blood lactate results rather from a fall in its hepatic utilization that from an increase in its production.