Administration Of Diethyl Maleate Research Articles

Renal and hepatic glutathione pool modifications in response to depletion treatments.

In this study we examined the response of the renal and hepatic glutathione (GSH) pool in rats to drastic GSH depletion treatments. For this purpose, we used a protein-free diet, starvation, and the injection of varying doses of diethyl maleate as depleting agents. We analysed GSH levels in both kidney and liver tissue homogenates after rats were fed a protein-free diet for 2 or 7 days or starved for 1, 2, or 3 days, as well as after diethyl maleate administration in a single maximal dose or in varying doses. The results indicated that the liver GSH pool was always more labile than the kidney GSH pool. Moreover, kidney GSH levels were almost unchanged after 7 days on a protein-free diet or after 2 days of starvation, while liver showed significant changes in GSH levels. When we analysed the repletion rate, kidney had higher kinetic parameters (k = 0.148 h-1) than liver (0.097 h-1). We conclude that efficient mechanisms of maintaining GSH levels exist in the kidney and these may serve to avoid GSH diminution and hence preserve renal function during states of GSH depletion.

Pharmacokinetic aspects of sulfobromophthalein transport after diethyl maleate pretreatment in rats.

The pharmacokinetics of sulfobromophthalein was studied in the rat after depletion of hepatic glutathione levels induced by intraperitoneal administration of diethyl maleate (4.0 mmol/kg). After an intravenous bolus injection of sulphobromophthalein (120 mumol/kg) a biexponential plasma decay was found both in control and diethyl maleate pretreated rats. The initial plasma clearance Cl12 was not modified by diethyl maleate administration. The rate constant of biliary excretion K23 was significantly lowered in diethyl maleate pretreated rats, which could by explained by the change in the biliary excretory process. The cumulative biliary excretion of sulphobromophthalein was decreased by about 50% following diethyl maleate injection, with a reduction of the percentage of conjugated dye excreted into bile.

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.

Effect of diethylmaleate and other glutathione depletors on protein synthesis

The α,β-unsaturated carbonyl compound diethylmaleate (DEM) depletes glutathione (GSH) from liver and other tissues, and for this reason it is often used in toxicological research to study the GSH-mediated metabolism of xenobiotics. In addition to GSH depletion, however, DEM has been shown to have other nonspecific effects, such as alteration of monooxygenase activities or glycogen metabolism. In this study we found that DEM (1 ml/kg) inhibited protein synthesis in brain and liver, following in vivo administration to mice. Protein synthesis was measured as the incorporation of [ 3H]valine into trichloroacetic acid-precipitable material. Administration of DEM also decreased body temperature by 2–3°. By increasing the environmental temperature from 22° to 35° the hypothermie effect of DEM was prevented, without affecting its ability to deplete GSH from brain and liver. Furthermore, when mice were maintained at 35°, DEM still caused a significant decrease in protein synthesis, suggesting that this effect was only partially due to hypothermia. To test whether inhibition of protein synthesis was related to GSH depletion, groups of animals were dosed with the α,β-unsaturated carbonyl phorone (diisopropylidenacetone) or the specific inhibitor of GSH synthesis, buthionine sulfoximine (BSO). Phorone decreased GSH in liver and brain; however, it had no effect on protein synthesis. BSO decreased GSH levels in liver and kidney, but not in brain, and did not have any effect on protein synthesis in any of these tissues, nor did it cause any hypothermia. Furthermore, when hepatic GSH content was decreased by in vivo administration of DEM or BSO, there was no inhibition of protein synthesis measured in vitro. These results indicate that, at the dose normally used to deplete GSH from various tissues, DEM also exerts an inhibitory effect on protein synthesis, which appears to be only partially due to its hypothermic effect, and is independent from GSH depletion. BSO, which, in our experimental conditions, lacks this and other nonspecific effects, might be a good alternative for studies aimed at characterizing the role of GSH in the metabolism and toxicity of chemicals.

Mechanism of protection by zinc against mercuric chloride toxicity in rats: effects of zinc and mercury on glutathionine metabolism.

To investigate the mechanism by which zinc suppresses mercury toxicity, the effects of zinc and mercury on glutathione (GSH) metabolism in the rat kidney were studied. When the time course of GSH level in the rat kidney was examined at 2, 6, and 12 h after treatment of rats with both metals, an increase of GSH was found and was apparently related to the activation of some GSH-associated enzymes. In the kidney of rats treated with both metals, the response of the protective function involving GSH and GSH-associated enzymes depended on the magnitude of mercury toxicity but appeared to be independent of the zinc dosage. The administration of diethyl maleate (DEM), which depletes GSH, increased lipid peroxidation and mercury toxicity concomitantly with a decrease of GSH level in the kidney of rats treated with zinc and mercury. In conclusion, the data suggest that an increased GSH level in the kidney resulting from the activation of GSH-associated enzymes plays a role in the protective effect of zinc against mercury toxicity.

Biliary excretion of glutathione and glutathione disulfide in the rat. Regulation and response to oxidative stress.

Regulation of the biliary excretion of reduced glutathione (GSH) and glutathione disulfide (GSSG) and responses to selected model toxins were examined in male Sprague-Dawley rats. In control and phenobarbital-pretreated rats in which the intrahepatic concentration of GSH was modulated by the administration of diethyl maleate or acetaminophen, the biliary concentration of GSH was consistently lower than, but directly proportional to, the intrahepatic concentration of GSH. Furthermore, increments in bile flow produced by the infusion of sulfobromophthalein (BSP)-glutathione were associated with proportional increases in the biliary excretion of GSH, suggesting that GSH passes into bile passively along a concentration gradient. In contrast, GSSG appears to be secreted into bile against a steep concentration gradient. An increased hepatic production and biliary excretion of GSSG resulted from the administration of t-butyl hydroperoxide. Measurement of biliary GSSG and BSP during a constant infusion of the GSH adduct of BSP indicated that GSSG shares a common excretory mechanism with GSH adducts. Diquat, nitrofurantoin, and paraquat also markedly stimulated the biliary excretion of GSSG. On a molar basis, these compounds generated much more GSSG than a direct substrate for glutathione peroxidase such as t-butyl hydroperoxide, indicating that the compounds undergo redox-cycling with concomitant production of hydrogen peroxide. Aminopyrine (0.8 mmol/kg) also significantly increased biliary GSSG. This increase, however, was associated with a proportional increase in bile flow and in the biliary excretion of GSH such that the GSSG/GSH ratio in bile did not change. Acetaminophen and chloroform, two compounds generating electrophilic metabolites that deplete intrahepatic GSH, led to a progressive decrease in the biliary excretion of GSH and GSSG. Furosemide and dimethylnitrosamine, the electrophilic metabolites of which do not deplete hepatic GSH, minimally altered biliary GSH and GSSG. Similarly, carbon tetrachloride and iproniazid, which yield organic radical metabolites that can peroxidize membrane lipids, did not increase the biliary excretion of GSSG. This finding indicates that membrane-bound lipid hydroperoxides may not be good substrates for glutathione peroxidases. The measurement of the biliary excretion of GSSG and of the GSSG/GSH ratio in bile is a sensitive index of oxidative stress in vivo and thus complements other in vivo parameters for the study of reactive intermediates of xenobiotics such as the determination of covalent binding, the formation of lipid hydroxy acids, and the depletion of intracellular GSH.

Investigations into the mechanism of coumarin-induced hepatotoxicity in the rat.

The administration of single doses of coumarin to the rat was found to produce hepatic centrilobular necrosis and also to depress a number of hepatic enzyme activities within 24 h. Coumarin-induced liver damage was diminished by pretreatment with cobaltous chloride but potentiated by the administration of diethyl maleate. Hepatic reduced non-protein sulphydryl levels were rapidly depleted following coumarin treatment whereas urinary mercapturic acid excretion was enhanced suggesting the formation of a coumarin metabolite or metabolites hitherto undetected in this species. In in vitro studies [3-14C]coumarin was converted by rat hepatic microsomes to reactive intermediates which became bound covalently to microsomal proteins. Additional studies established that the formation of reactive metabolites was a cytochrome P-450 dependent process and that macromolecular binding could be inhibited by sulphydryl compounds (including reduced glutathione) and hepatic cytosol fractions. These results demonstrate that coumarin-induced hepatotoxicity in the rat is likely to be mediated via one or more reactive metabolites generated by cytochrome P-450 dependent enzymes and that reduced glutathione and other thiol agents constitute a detoxification pathway.

Effect of adrenalectomy, pretreatment with SKF 525-A, phenobarbital and diethyl maleate on the acute toxicity of fenitrothion in male rats.

The effect of adrenalectomy (Adx), SKF 525-A, phenobarbital (PB), and diethyl maleate (DEM) on the acute toxicity of fenitrothion was investigated in male rats by assessing the degree of plasma cholinesterase activity. PB, 60 mg/kg/day for 3 days, exerted no protective effect on the toxicity of fenitrothion (100 mg/kg, p.o.) given 24 h after the last injection. In adrenalectomized and SKF 525-A-pretreated rats, the toxicity of fenitrothion was lower than that of the controls. Fenitrothion toxicity was increased by administration of DEM (1 ml/kg), which depletes hepatic glutathione (GSH) levels. In vitro, the rates of fenitrothion decomposition and fenitrooxon formation by microsomes were markedly affected by PB, SKF 525-A and Adx. The decomposition of fenitrooxon by the microsomal fraction and GSH-dependent decomposition of fenitrooxon by the soluble fraction were not affected by PB, SKF 525-A and Adx pretreatment. The GSH-dependent decomposition of fenitrothion and fenitrooxon was increased by addition of GSH to the incubation mixture. The present results indicate that the GSH-dependent metabolic pathway plays an important role in the detoxication of fenitrothion.

Studies on hydrazine hepatotoxicity. 2. Biochemical findings.

Hydrazine causes a dose-related increase in liver triglycerides and in liver weight and causes a decrease in hepatic glutathione. The threshold dose for the toxic effect is around 10 mg/kg, and the optimal effect is seen after a dose of 40 mg/kg. The effect of hydrazine on liver weight and glutathione was detectable within 30 min of dosing, but the elevation of hepatic triglycerides was not detectable until 4 h after dosing. At 24 h after a dose of 60 mg hydrazine/kg, hepatic reduced glutathione was approximately 50% of the control value and triglycerides were about 7 times the normal level. In vitro studies indicated that hydrazine is metabolized by rat liver microsomal enzymes, this being dependent on NADPH and oxygen. Pretreatment of animals with phenobarbital or piperonyl butoxide respectively decreases and increases the hepatotoxicity. Prior depletion of hepatic glutathione by administration of diethyl maleate had no effect on the toxicity. Pyruvate azine, a probable metabolite of hydrazine, is much less toxic than hydrazine itself on a molar basis. These and other results suggest that although hydrazine is metabolized via several routes, the hepatotoxicity may well be due to the parent compound rather than a metabolite.

Changes of the Golgi apparatus induced by diethylmaleate in rat hepatocytes.

Ultrastructural studies of the liver of rats given diethylmaleate (DEM) (0.7 ml per kg body, i.p.) weight revealed marked alterations of the configuration of the Golgi complex in all hepatocytes. This consisted in dilatation of the cisternae leading to the formation of large cysts. The alteration was more prominent 15 min after administration of the drug, coincident with a 2-fold increase of bile flow and a decrease of hepatic glutathione to 15% of controls. The morphology returned to normal at 45 min after DEM administration together with a return of bile flow to control values whereas hepatic glutathione level remained markedly depressed for 2 hr, rising slowly afterwards. No other signs of altered hepatic morphology developed up to 5 hr. The morphological changes correlated with altered bile flow induced by DEM, suggesting involvement of the Golgi complex in biliary excretion of osmotically active glutathione conjugates of DEM.

Effect of allylisopropylacetamide on glutathione metabolism in the rat liver. The possible role of glutathione in the induction of 5-aminolaevulinate synthase.

Administration of allylisopropylacetamide to rats caused a marked decline in the concentrations of reduced and oxidized glutathione in the liver. However, this decrease occurred in the presence of uninhibited activities of gamma-glutamylcysteine synthase and glutathione reductase, and unaltered activities of glutathione transferases A, B and C. The administration of cysteine, the rate-limiting precursor of glutathione formation, to rats treated with allylisopropylacetamide potentiated the inductive effects of the agent on 5-aminolaevulinate synthase, and markedly decreased the extent of decrease in glutathione concentrations by the agent. Conversely, the administration of diethyl maleate, which depletes the hepatic glutathione concentrations, to allylisopropylacetamide-pretreated rats (1h) diminished the extent of 5-aminolaevulinate synthase induction and the production of porphyrins by nearly 50%, when measured at 16h. This treatment did not alter the extent of non-enzymic degradation of liver haem by allylisopropylacetamide. When diethyl maleate was administered to the animals possessing high 5-aminolaevulinate synthase activity (at 3, 7 and 15h after allylisopropylacetamide), in 1h the enzyme activity was markedly decreased. Diethyl maleate had no effect on induction of 5-aminolaevulinate synthase by 3,5-diethoxycarbonyl-1,4-dihydrocollidine, also a potent porphyrinogenic agent. Diethyl maleate alone neither inhibited 5-aminolaevulinate synthase activity nor decreased the cellular content of porphyrins and haem. The data suggest that the decreases observed in the glutathione concentrations after allylisopropylacetamide administration are not the result of decreased production of the tripeptide. Rather, they most likely reflect the increased utilization of glutathione. The findings further suggest that the inhibition by diethyl maleate of allylisopropylacetamide-stimulated 5-aminolaevulinate synthase involves the inhibition of induction processes.

Depression of cytochrome P-450-dependent drug biotransformation by adriamycin

The cytochrome P-450-dependent mixed function oxidase system was depressed following the administration of adriamycin. Cytochrome P-450, aminopyrine N-demethylase, and benzo( a)pyrene hydroxylase were significantly decreased in hepatic microsomes prepared from rats treated with a single dose of adriamycin (10 mg/kg subcutaneously) 4 days previously. Total microsomal protein levels and cytochrome b 5 levels remained unchanged. The administration of cysteamine 1 hr prior to adriamycin prevented the loss of the cytochrome P-450 and the decrease in drug biotransformation. The administration of diethylmaleate had no effect on the ability of adriamycin to decrease this enzyme system. The loss of drug biotransformation and the protection offered by cysteamine is correlated with the lipid peroxidation activity in hepatic microsomes. This suggests that the loss of cytochrome P-450 and related drug biotransformation is related to the generation of free radicals and subsequent lipid peroxidation in the liver.

Protective effect of diethylmaleate pretreatment on carbon tetrachloride hepatotoxicity

The administration of diethylmaleate (DEM) to fed and fasted rats produced a marked depletion of hepatic reduced glutathione (GSH) to 26 and 20% of respective saline control values 30 min after drug administration. When carbon tetrachloride (CCl 4) was administered to fasted animals pretreated with saline or DEM, the serum enzymes GOT, GPT, and ICDH were elevated 24 hr after intoxication, but the increases were greater in the saline-pretreated group ( p < 0.05). The administration of CCl 4 to fed animals produced elevations in serum GOT, GPT, and ICDH 24 hr after intoxication that were of similar magnitude in the saline- and DEM-pretreated groups ( p > 0.05). CCl 4 administration to fed rats produced altered hepatocellular architecture that was of similar magnitude in saline- and DEM-pretreated animals. However, in fasted animals, the histological changes following CCl 4 administration were more severe in saline-pretreated animals when compared to the DEM-pretreated group. The data suggest that DEM pretreatment exerts a protective effect against CCl 4-induced hepatic injury in fasted rats that may be related to the ability of DEM to inhibit hepatic microsomal drug metabolism.

8] Chemical depletion of glutathione in vivo

Publisher Summary The method for lowering tissue glutathione (GSH) levels involves the administration of compounds that react enzymically with GSH to form conjugates. The inhibition of GSH synthesis can also result in the depletion of this tripeptide in the organs with a sufficient turnover rate. The conversion of GSH to its oxidized form, glutathione disulfide (GSSG), has also been used to deplete GSH, mainly in isolated cell preparations; the choice of the depleting agent depends on the type of system under study. Diethyl maleate (DEM) remains one of the most useful compounds for depleting hepatic GSH in vivo. Intraperitoneal administration of DEM reduces the hepatic GSH levels of rats to 6-20% of control values in 30 min and for a period of 2 to 4 hr. Phorone is another α,β-unsaturated carbonyl compound that may be useful as a GSH depleting agent. As in the case of DEM, the conjugation of phorone with GSH is catalyzed by glutathione transferases.

Induction of paracetamol metabolism in the isolated perfused kidney.

1. At a perfusate concn. of 3.5-4.0 mM, 59 plus or minus 9 nmol of paracetamol h per g wet wt. were oxidized by isolated rat kidney. 2. Approx. half the paracetamol undergoing oxidation was converted to a mercapturic acid metabolite and the remainder was covalently bound to kidney protein. 3. Addition of GSH to the perfusate decreased the level of covalent binding. Depletion of cellular GSH, by prior administration of diethyl maleate, significantly decreased formation of the mercapturic acid metabolite. 4. The metabolic pathways of glucoronylation, sulphation and mercapturic acid formation were induced either by 3-methylcholanthrene pretreatment or by prolonged feeding of aspirin or paracetamol; covalent binding of paracetamol to kidney protein was not increased.

Effect of fasting, diethyl maleate, and alcohols on carbon tetrachloride-induced hepatotoxicity

The effect of fasting, diethyl maleate treatment, and methanol, ethanol, iso-propanol, or tert-butanol treatment on carbon tetrachloride (CCl 4)-induced hepatotoxicity in male Sprague-Dawley rats was studied. A 24-hr fast and the administration of diethyl maleate were found to be equipotent and not additive in potentiating CCl 4-induced hepatotoxicity; this potentiation appeared to be due to a depletion of hepatic glutathione (GSH) levels. Concomitant diurnal variations in hepatic GSH content and in CCl 4-induced hepatotoxicity also suggested hepatic GSH involvement in CCl 4-induced hepatic damage. Whereas ethanol has been reported to potentiate CCl 4-induced hepatotoxicity and to lower hepatic GSH levels, the present study suggested that these effects are due to an ethanol-induced loss of body weight. Methanol, iso-propanol, and tert-butanol, on the other hand, show the same maximal potentiation of CCl 4-induced hepatotoxicity and do so without inducing a depletion of hepatic GSH content or producing a loss of body weight. In contrast to a previous report, however, methanol was found to be markedly less effective on an equimolar basis than either iso-propanol or tert-butanol in potentiating CCl 4-induced hepatotoxicity. The study suggests that GSH is an important modulator of CCl 4-induced hepatotoxicity and also suggests that methanol, iso-propanol, and tert-butanol, but not ethanol, potentiate CCl 4-induced hepatotoxicity by a mechanism that does not involve altered hepatic GSH levels.

Effect of metabolic inhibitors, diethylmaleate and carbon tetrachloride-induced liver damage on glutathione S-trans-ferase activities in rat liver

Summary The effects of some antihepatotoxic agents on the glutathione content and the glutathione transferase activities in rat liver cytosol were studied. Dithiocarb, a potent metabolic inhibitor, had no effect on either parameter. (+)-cyanidanol-3 and dimethylsulfoxide (DMSO) were both well able to inhibit the glutathione transferase activity towards the epoxide substrate (1,2-epoxy-3-(p-nitrophenoxy)propane), but not that towards the aryl substrate (1-chloro-2,4-dinitrobenzene). This last activity was elevated after treatment with DMSO and following administration of Diethylmaleate which is a glutathione depleting agent and which exerts variable effects on drug-metabolizing enzymes. Liver injury by CCl 4 results in a gradual decrease in the enzymic activities, which lasts even 72 h after treatment and seems to be a consequence of liver cell regeneration associated with transient loss of cell differentiation. Again, the epoxide transferase activity seems to be more susceptible to factors inducing changes of enzymic activity.

Urinary excretion of mercapturic acids in chimpanzees and rats

The urinary excretion of mercapturic acids has been considered as an indicator for human exposure to environmental chemicals. To evaluate this concept, the excretion of urinary mercapturic acids was determined in chimpanzees and rats after the oral administration of single doses of naphthalene and diethylmaleate. The excretion rate of endogenous thioethers in the urine of untreated chimpanzees and rats was 18.0 ± 1.1 and 94.4 ± 2.8 μmol/kg/24 hr respectively. The value for man was nearly the same as found in chimpanzees. After the administration of naphthalene (30, 75, and 200 mg/kg) a dose-dependent increase of the excretion rate of urinary mercapturic acids up to 408 μmol/kg/24 hr was observed in the rat. In the chimpanzees the same treatment failed to increase the urinary thioether concentrations. The administration of diethylmaleate (30, 75, and 200 mg/kg) led to a dose-dependent increase in the excretion of urinary mercapturic acids in both species. In rats this increase was about twice that of chimpanzees. The results suggest that the chimpanzee is a relevant model for man to study the urinary excretion of mercapturic acids deriving from electrophilic compounds, whereas the rat is not. Experiments with [ 14C]-naphthalene indicate that the species differences observed are due to differences in the glutathione conjugation.

Acute paracetamol intoxication of starved mice leads to lipid peroxidation in vivo

Lipid peroxidation was monitored in female mice in vivo by the measurement of exhalated hydrocarbons. In liver homogenates in vitro lipid peroxidation as determined by malondialdehyde formation, and hepatic total glutathione levels were measured. After a dose of 500 mg/kg i.p. of paracetamol, the hepatic glutathione of fed mice decreased from 61 nmoles/mg liver protein to 30 nmoles/mg, while the animals expired 5 nmoles of ethane/kga · hr. The same dose in starved mice led to a glutathione level of 6 nmoles/mg and an exhalation rate of 125–150 nmoles ethane/kg · hr. In vivo determined and post-mortem in vitro determined lipid peroxidation correlated with a coefficient of 0.66. If hepatic glutathione was depleted to the same extent by administration of diethylmaleate, no significant lipid peroxidation was found. Our findings demonstrate that the drug-induced depletion of liver glutathione leads in vivo to lipid peroxidation, provided that the glutathione level has been diminished by starvation. The data indicate that glutathione depletion alone by other mechanisms does not account for lipid peroxidation. Hence the hepatoprotective role of liver glutathione against drug-induced liver injury has to be reconsidered in detail. This investigation shows a suitable model for such studies.

Effects of diethyl maleate on aryl hydrocarbon hydroxylase and on 3-methyl-cholanthrene-induced skin tumorigenesis in rats and mice.

Topical administration of diethyl maleate (DEM) and L-methionine sulfoximine (MS) reduced the L-glutathione (GSH) levels in kidneys, livers, and skin of inbred BALB/c mice. Topical administration of DEM to BALB/c mice also increased the latency period before development of skin tumors that were induced by 3-methylcholanthrene painting. Similar treatment with MS also increased the latency period, though the delay was not as striking as that observed after DEM administration. Furthermore, DEM, which was believed to be specific in its action in reducing tissue GSH, was also capable of inhibiting aryl hydrocarbon hydroxylase (AHH) both in vitro and in vivo. Cyclohexene sulfide, another "specific" inhibitor of GSH transferase, inhibited AHH activity as well. Accordingly, the blockade of AHH by DEM may have been partly responsible for the increased latency time in the skin tumorigenesis experiments.