Effect of phosphate transporter and methylation inhibitor drugs on the disposition of arsenate and arsenite in rats.

Arsenate (AsV) is biotransformed into the more toxic arsenite (AsIII) and monomethylarsonous acid (MMAsIII), but it is unknown how to decrease production of these harmful metabolites. We investigated the effects of foscarnet and fosfomycin, drugs interacting with the phosphate transporter, on biotransformation of AsV, an analog of inorganic phosphate. The effects of entacapone, an inhibitor of catechol-O-methyl transferase (COMT), and nitrous oxide, an inactivator of methylcobalamin, were also tested on the formation of MMAsIII from AsIII in order to clarify the role of COMT and methylcobalamin in biomethylation of AsIII. Arsenic in bile and urine of control and treated rats receiving AsV or AsIII was speciated by HPLC-HG-AFS. In AsV-injected rats, foscarnet, but not fosfomycin, increased the urinary excretion of AsV and decreased the biliary and urinary excretion of AsIII as well as biliary excretion of MMAsIII. In AsIII-injected rats, however, foscarnet failed to influence the excretion of AsIII and its metabolites, suggesting that this drug inhibits the hepatic uptake and renal reabsorption of AsV, thereby decreasing formation of AsIII and MMAsIII from AsV. Entacapone or nitrous oxide pretreatment slightly or not at all influenced the biliary excretion of MMAsIII and urinary excretion of dimethylarsinic acid (DMAsV) in AsIII-injected rats. In contrast, periodate-oxidized adenosine, an inhibitor of S-adenosylmethionine-dependent methyltransferases, nearly abolished appearance of methylated arsenic metabolites in bile and urine. Thus, foscarnet facilitates urinary clearance of AsV and decreases formation of toxic AsIII and MMAsIII, indicating that this drug may be used to promote elimination and counter toxification of AsV. Because entacapone and nitrous oxide influenced the excretion of MMAsIII and DMAsV negligibly, neither COMT nor methylcobalamin appears to be involved in arsenic methylation in rats.

Effects of methylmercury and organic acid mercurials on the disposition of exogenous selenium in rats.

Interaction of methylmercury (MM), an environmental and industrial toxicant, with selenium is well known but incompletely understood. Therefore, the effects of MM (10 micromol/kg i.v.) on the disposition of exogenous selenium were compared with those of other organic mercurials (merbromine, mercuribenzene sulfonic acid, and mercuribenzoic acid) in anesthetized bile duct-cannulated rats injected with sodium [(75)Se]selenite (10 micromol/kg i.v.). The mercurial organic acids (10 micromol/kg i.v.) differed strikingly from MM in their influence on selenium disposition. They promoted renal and hepatic accumulation as well as biliary excretion of selenium but decreased distribution to the muscle, testis, and brain as well as the pulmonary excretion of selenium. In contrast, MM altered selenium distribution in an opposite fashion: it diminished the biliary output of selenium and enhanced selenium exhalation. GC-MS analysis verified that this latter paradoxical effect resulted from increased exhalation of dimethyl selenide. Further studies indicated that the MM-induced increase in pulmonary excretion of dimethyl selenide cannot be due to a diminished conversion of this volatile selenium compound to trimethylselenonium ion (TMSe(+)), because MM influenced neither the urinary excretion nor the hepatic and renal concentration of TMSe(+) in selenite-injected rats. Compared to the selenite-exposed rats, the selenite plus MM-injected animals exhibited a significant rise in the hepatic level of S-adenosylmethionine (SAME), the endogenous methyl donor in selenium methylation, and the ratio of SAME to S-adenosylhomocysteine. Based on these and others' observations, it is hypothesized that MM may increase hepatic availability of SAME in selenite-dosed rats by counteracting selenite-induced inactivation of SAME synthetase, thereby facilitating SAME synthesis, and/or by acting as a methyl donor in formation of dimethyl selenide, thereby sparing SAME. In summary, the toxicologically and ecologically relevant interaction of MM and selenite is not mimicked by organic acid mercurials, possibly because it results in formation of lipophilic Hg- and Se-containing common compound(s) and because it also appears to involve methyl transfer from MM to selenium.

Effects of arsenic-, platinum-, and gold-containing drugs on the disposition of exogenous selenium in rats.

Having found that the electrophilic model compound sulfobromophthalein markedly altered the fate of exogenous selenium in the body by reacting in vivo with nucleophilic selenium metabolites, the effects of metal-containing drugs with expected selenium reactivity were tested on biliary, urinary, and pulmonary excretion. Tissue distribution of selenium in selenite-injected rats was also examined. Coadministration with [(75)Se]selenite (10 micromol/kg, iv) of the trypanosomicid arsenicals (100 micromol/kg, iv) trimelarsan (TMA) or melarsoprol (MAP), the antitumor cisplatin (25 micromol/kg, iv), or the antirheumatic gold sodium thiomalate (25 or 50 micromol/kg, iv) significantly altered the disposition of (75)Se, whereas carboplatin (100 micromol/kg, iv) did not produce such an effect. The most dramatic alterations included the approximately 20-fold increase in the biliary excretion rate of selenium in response to TMA and MAP, the almost complete cessation of the exhalation of selenium as dimethyl selenide after administration of the arsenic- and gold-containing drugs, and the manifold accumulation of selenium in the blood plasma following gold injection. Direct chemical reaction of the drugs with nucleophilic selenite metabolites in the body may underlie these alterations. The tight coordination in time and extent observed between the biliary excretion of arsenic and selenium in rats receiving either of the arsenicals and selenite supports this hypothesis. However, attempts to detect selenium-containing biliary metabolites of TMA and MAP have failed, possibly owing to their instability. In summary, the arsenic-, platinum- and gold-containing drugs significantly influence the fate of exogenous selenium, whereby they may adversely affect the availability of this essential element for synthesis of selenoenzymes. Furthermore, the capability of TMA and MAP to enhance the biliary and total excretion of selenium renders these drugs significant candidates for antidotes in selenium intoxication.

Biliary and urinary excretion of inorganic arsenic: monomethylarsonous acid as a major biliary metabolite in rats.

In rats exposed to arsenite (AsIII) or arsenate (AsV), the biliary excretion of arsenic depends completely on availability of hepatic glutathione, suggesting that both AsIII and AsV are transported into bile in thiol-reactive trivalent forms (Gyurasics et al. [1991], Biochem. Pharmacol. 42, 465-468). To test this hypothesis, the bile and urine of bile duct-cannulated rats injected with AsIII or AsV (50 micromol/kg, iv) were collected periodically for 2 h and analyzed for arsenic metabolites by HPLC-hydride generation-atomic fluorescence spectrometry. Arsenic was excreted predominantly into bile in AsIII-injected rats, but the urine was the main route of excretion in AsV-exposed rats. Injected AsIII was excreted in urine practically unchanged, whereas both AsV and AsIII appeared in urine after administration of AsV. Irrespective of the arsenical administered, the bile contained 2 main arsenic species, namely AsIII and a hitherto unidentified metabolite. Formation of this metabolite could be prevented by pretreatment of the rats with the methylation inhibitor periodate-oxidized adenosine, indicating that it is a methylated arsenic compound. This metabolite could be converted in vitro into monomethylarsonic acid (MMAsV) by oxidation, whereas synthetic MMAsV could be converted into the unknown metabolite by reduction. Consequently, this biliary metabolite of both AsIII and AsV is monomethylarsonous acid (MMAsIII), a long-hypothesized, but never identified, intermediate in the biotransformation of AsIII and AsV. Although MMAsIII is thought to be formed from an oxidized precursor, rats injected with MMAsV did not excrete MMAsIII. In summary, the inorganic arsenicals investigated are transported into bile exclusively in trivalent forms, namely as AsIII and MMAsIII, but are excreted in urine in both tri- and pentavalent forms. Identification of MMAsIII is signified by the fact that this metabolite is more toxic than AsIII and AsV and thus formation of MMAsIII represents toxification of inorganic arsenic.

Role of glutathione in the biliary excretion of the arsenical drugs trimelarsan and melarsoprol.

After administration of the inorganic sodium arsenite or arsenate to rats, the biliary excretion of arsenic is rapid, is accompanied by the biliary output of large amounts of GSH, and is completely arrested by the GSH depletor diethyl maleate (DEM). We studied the biliary excretion of trimelarsan (TMA) and melarsoprol (MAP) in rats in order to determine whether biliary excretion is also significant in the disposition of these trivalent organic arsenicals that are used as therapeutic agents and whether GSH is also involved in their hepatobiliary transport. After injection of either drug (100 micromol/kg, i.v.), arsenic was rapidly excreted in bile (up to 1 micromol/kg. min, approximately 55% of dose/100 min). Concurrently, TMA and MAP increased the biliary output of GSH 3- and 6 fold, and lowered the hepatic GSH content by 24% and 27%, respectively. In TMA-injected rats, pretreatment with DEM or buthionine sulfoximine decreased the initial biliary excretion of arsenic by 75% and 40%, respectively, whereas in MAP-injected rats these GSH depletors diminished arsenic output by 45% and 20%. Both arsenicals reacted with GSH in vitro, giving rise to the same product, which was also shown by HPLC analysis to be a major biliary metabolite of both TMA and MAP. This metabolite was sensitive to gamma-glutamyltranspeptidase in vitro and its biliary excretion was virtually prevented by the GSH depletors, confirming that it is a GSH conjugate (purportedly melarsen-diglutathione). Some TMA was excreted in the bile unchanged, whereas a significant amount of MAP also appeared there as two glucuronides. The biliary excretion of unchanged TMA and MAP glucuronides was increased by experimental depletion of GSH. These studies indicate that the biliary excretion of TMA and MAP (1) is very significant in their disposition, (2) is partially dependent on the hepatic availability of GSH, as these arsenicals are excreted in part as a GSH conjugate, and (3) is concomitant with the increased appearance of GSH in bile, probably originating from dissociation of the unstable GSH conjugate of these arsenicals. Thus, conjugation with GSH is important in the elimination of both TMA and MAP, although glucuronidation is also involved in the fate of MAP.

Effect of chlorophenoxyacetic acid herbicides on glycine conjugation of benzoic acid.

1. 2,4-Dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) (0.1-0.5 mmol/kg i.p.) delayed the disappearance of injected benzoate from blood and diminished the urinary excretion of the formed benzoylglycine, but elevated the blood levels of benzoylglycine in rat, suggesting that these herbicides interfere with both the formation and the renal transport of benzoylglycine. 2. Inhibition of the renal excretion of benzoylglycine by 2,4-D or 2,4,5-T (0.5 mmol/kg i.p.) was directly demonstrated in rat injected with benzoylglycine. 3. Inhibition of benzoylglycine formation from benzoic acid by 2,4-D or 2,4,5-T (0.5 mmol/kg i.p.) was directly demonstrated in renal pedicles-ligated rats injected with benzoate. 4. Neither 2,4-D nor 2,4,5-T influenced the hepatic concentrations of ATP, coenzyme A (CoA) or glycine; therefore, it is unlikely that they inhibit glycine conjugation of benzoic acid by diminishing the availability of co-substrates. 5. Although the chlorophenoxyacetic acids did not appear to be a substrate for the mitochondrial acyl-CoA synthetases, both 2,4-D and 2,4,5-T diminished the activity of benzoyl-CoA synthetase (but not that of benzoyl-CoA:glycine N-acyltransferase) in solubilized hepatic mitochondria. These findings suggest that 2,4-D and 2,4,5-T impair benzoylglycine formation in rat by inhibiting benzoyl-CoA synthetase.

Role of glutathione and methylation in the biliary excretion of selenium. The paradoxical effect of sulfobromophthalein.

Biotransformation of selenite involves both reactions with GSH and methylations. Therefore, the role of GSH, methylation, and the hepatobiliary GSH transporter was investigated in the biliary excretion of selenium in rats injected with sodium [75Se]selenite (1-10 micromol/kg, i.v.). Biliary output of selenium exhibited an apparent capacity limitation with an approximately 3 nmol/kg x min maximal rate and a dose-related decline in the fractional excretion. HPLC analysis of bile indicated absence of selenite and presence of selenodiglutathione (GS-Se-SG) and/or its hydrolysis products as the major biliary selenite metabolites. Depletion of hepatic glutathione by D,L-buthionine-[S,R]-sulfoximine or diethyl maleate decreased selenium excretion into bile by 60 and 80%, respectively. In contrast, inhibitors of methylation, i.e. periodate-oxidised adenosine or ethionine doubled the rate of biliary selenium excretion. While indocyanine green--an inhibitor of hepatobiliary GSH transport--failed to influence biliary selenium output, sulfobromophthalein (BSP)--another inhibitor of this sort--dramatically enhanced it. This effect was found to be a function of the dose of both selenite and BSP. The degree of BSP-induced enhancement of the selenium excretion rate gradually increased with elevation of the selenite dose, approaching 20-fold at 10 micromol/kg selenite. In contrast, the stimulatory effect of BSP on biliary selenium output was maximal at 50-100 micromol/kg and gradually lessened with elevation of the BSP dose above 100 micromol/kg. In summary, this study revealed that the biliary excretion of selenium depended on availability of hepatic GSH, probably for formation of GS-Se-SG, the putative cholephilic selenite metabolite. Methylation counteracted selenium excretion into bile and thus may contribute to the apparent capacity limitation of biliary selenium excretion. Finally, selenium output into bile was insensitive to inhibitors of the hepatobiliary GSH transporter, and was enhanced, paradoxically, by BSP several-fold. The mechanism of this unexpected effect is explored in the adjoining article.

Enhancement of selenium excretion in bile by sulfobromophthalein: elucidation of the mechanism.

This work was intended to explore the mechanism whereby sulfobromophthalein (BSP), an electrophilic and cholephilic organic acid, increases the biliary excretion of selenium in rats injected with sodium [75Se]selenite. In such animals, neither BSP-glutathione conjugate nor dibromosulfophthalein, nonelectrophilic congeners of BSP, enhanced the hepatobiliary transport of selenium, suggesting that reaction of nucleophilic selenite metabolites formed in vivo with the injected BSP may be involved. Indeed, HPLC analysis of bile from rats receiving [75Se]selenite and BSP revealed two peaks (X and Y) that were simultaneously detected both by absorbance as BSP metabolites and by radioactivity as [75Se] metabolites, indicating that these represent selenium-containing BSP metabolites. Pretreatment of rats with inhibitors of selenium methylation, such as periodate-oxidized adenosine (PAD) and ethionine, drastically diminished the size of peak X, while increasing (PAD) or not influencing (ethionine) the size of peak Y. This finding indicates that production of metabolite X, but not Y, is dependent on formation of methylated selenium metabolites. A compound chromatographically indistinguishable from that in peak X was formed in vitro during incubation of BSP with methylselenol, suggesting that biliary metabolite X is identical to the reaction product of BSP and selenite-derived methylselenol. Incubation of BSP with selenite in the presence of a thiol, namely glutathione, cysteine or N-acetylcysteine (which convert selenite into nucleophilic products, i.e. the respective selenopersulfides and hydrogen selenide) resulted in product(s) chromatographically identical to the biliary selenium-containing BSP metabolite(s) of peak Y, irrespective of the nature of the thiol used. Thus, biliary metabolite(s) Y may be reaction products of BSP and hydrogen selenide. Finally, BSP significantly diminished exhalation of dimethyl selenide in selenite-injected rats, purportedly because it reacted with precursors of dimethyl selenide, that include hydrogen selenide and methylselenol. In summary, BSP increases biliary excretion of selenium in rats receiving selenite because it forms selenium-containing BSP metabolites that are readily transported into bile. It is suggested that the in vivo reaction of nucleophilic selenite metabolites with electrophilic compounds may influence the fate of selenium and may contribute to some of the effects of this essential and anticarcinogenic metalloid.

[Human drug metabolizing enzymes. II. Conjugation enzymes]. / Human gyógyszermetabolizáló enzimek. II. Konjugációs enzimek.

In this review we focus on human conjugation enzymes (UDP-glucuronyltransferases, methyl-trasferases, N-acetyl-transferases, O-acetyl-transferases, Amidases/carboxyesterases, sulfotransferases, Glutation-S-transferases and the enzymes involved in the conjugation with amino acids) that participate in the metabolism of xenobiotics. Although conjugation reactions in most of the cases result in detoxication, more and more publications prove that the reactions catalysed by these enzymes very often lead to activated molecules that may attack macromolecules (proteins, RNAs, DNAs), resulting in toxicity (liver, neuro-, embryotoxicity, allergy, carcinogenecity). We have summarised the data available on these enzymes concerning their catalytic profile and specificity, inhibition, induction properties, their possible role in the generation of toxic compounds, their importance in clinical practice and drug development.

Effects of fibrates on the glycine conjugation of benzoic acid in rats.

In the course of glycine conjugation, benzoic acid is successively converted into benzoyl-CoA and benzoylglycine by mitochondrial enzymes (i.e. benzoyl-CoA synthetase and benzoyl-CoA/glycine N-acyltransferase, respectively), utilizing ATP, CoA, and glycine. Large doses of benzoate deplete CoA from the liver, suggesting that the supply of CoA may limit the capacity for glycine conjugation. Because fibrates are known to increase hepatic CoA synthesis, we examined whether treatment with fenofibrate or bezafibrate enhanced the capacity of rats to conjugate benzoic acid with glycine. Dietary administration of fenofibrate or bezafibrate (2.5 mmol/kg of feed, for 10 days) increased hepatic CoA levels 8-10-fold, while not affecting hepatic ATP levels; only fenofibrate elevated, albeit moderately, the concentration of glycine in liver. Hepatic mitochondria isolated from fibrate-fed rats, compared with those from controls, exhibited unchanged benzoyl-CoA synthetase activity but higher benzoyl-CoA hydrolase and lower benzoyl-CoA/glycine N-acyltransferase activities. Feeding with either fibrate increased liver mass by 50-60%. Control and fibrate-fed rats were administered benzoate at different doses, one to produce a large demand for CoA (i.e. 2 mmol/kg, iv) and two others to produce smaller demands for CoA (i.e. 1 mmol/kg or 2 mmol/kg plus glycine, iv). Fenofibrate-fed rats, and to a lesser extent bezafibrate-fed animals, exhibited increased glycine conjugation capacity, as indicated by faster disappearance of benzoate from the blood and appearance of benzoylglycine in the blood and urine, compared with controls; however, fibrates were not more effective in rats receiving the benzoate dose that produced the greatest demand for CoA. In contrast, benzoylglycine formation from benzoate (0.1-1 mM) was not enhanced in liver slices from fibrate-fed rats; moreover, it was lower than control levels in slices from bezafibrate-fed animals. Bezafibrate, but not fenofibrate, given to rats in a single dose (0.5 mmol/kg, ip) decreased the elimination and glycine conjugation of benzoate, indicating that bezafibrate is a direct inhibitor of glycine conjugation. In summary, fibrates influence glycine conjugation in a complex manner. Some fibrate-induced alterations (i.e. increased benzoyl-CoA hydrolase and decreased glycine transferase activities and direct inhibition by bezafibrate) can potentially hinder conjugation of benzoate with glycine, thus precluding conclusions regarding whether increased CoA availability enhances glycine conjugation. Fibrate-induced hepatomegaly appears to significantly contribute to the increased glycine conjugation capacity of rats treated with fenofibrate or bezafibrate.

Interactions between selenium and group Va-metalloids (arsenic, antimony and bismuth) in the biliary excretion.

The interrelationship between the biliary excretion of exogenous group Va-metalloids (arsenic, antimony and bismuth) and selenium, as well as endogenous glutathione has been studied in rats injected intravenously with sodium selenite and one of the group Va-metalloids. Arsenic, antimony and bismuth appeared in the bile of rats together with large amounts of non-protein thiols (NPSH, representing glutathione and its SH-containing degradation products) and, with the exception of bismuth, they caused choleresis. Significant interactions were observed in the hepatobiliary disposition between selenium and each of the group Va-metalloids, however, their outcomes were not uniform. When coadministered with sodium arsenite or arsenate, selenite enhanced the initial biliary excretion of arsenic 2- and 8-fold, respectively, without further increasing the concomitant excretion of NPSH or the choleretic effect of arsenicals. However, selenite augmented neither the excretion of antimony or bismuth, nor the simultaneous biliary release of NPSH. In turn, arsenite, arsenate and antimony potassium tartrate increased the initial biliary excretion of selenium more than 10-fold and enhanced the accumulation of selenium in blood (exclusively in the erythrocytes). In contrast, administration of bismuth ammonium citrate diminished both the biliary excretion and the erythrocytic accumulation of selenium, while causing retention of selenium in the blood plasma. In rats receiving arsenic or antimony with selenite, the time courses of the biliary excretion of these group Va-metalloids, selenium and NPSH were similar. It is hypothesised that incorporation of selenol metabolites of selenite into the glutathione complexes of arsenic and antimony, resulting in cholephilic ternary complexes, accounts for the arsenic- and antimony-induced augmentation of the hepatobiliary transport of selenium. However, additional chemical and/or dispositional mechanisms are thought to be responsible for the selenite-induced increase in biliary excretion of arsenic.

Does hepatic ATP depletion impair glycine conjugation in vivo?

Conjugation with glycine, a reaction important in the elimination of carboxylic acids (e.g. benzoic and salicylic acids), takes place in hepatic mitochondria and uses ATP, coenzyme A, and glycine. Although normal ATP supply does not limit glycine conjugation in vivo (Gregus, Z., et al., Drug Metab. Dispos. 20, 234, 1992), ATP deficiency may impair it. This hypothesis was tested by examining the effect of ATP depletors (oligomycin, fructose, and ethionine) on glycine conjugation and elimination of benzoic acid in rats. Pretreatment with the mitochondrial ATP synthesis inhibitor oligomycin (0.5-2 mg/kg, intraportally) decreased glycine conjugation of benzoic acid markedly and in a dose-dependent manner, as indicated by the delayed elimination of benzoate and delayed appearance of benzoylglycine in blood. Oligomycin also dramatically diminished urinary excretion of benzoylglycine, because it inhibited not only formation of benzoylglycine from benzoate, but also the renal transport of benzoylglycine. Treatment with fructose (a consumer of both cytosolic and mitochondrial ATP) or ethionine (a consumer of cytosolic ATP) depleted hepatic ATP from approximately 2.5 micromol/g to levels comparable with those observed after administration of 1 mg/kg oligomycin (approximately 1.2 micromol/g). Despite this, elimination of benzoate and formation of benzoylglycine were decreased less by fructose than by oligomycin and only negligibly by ethionine. ATP depletors did not influence hepatic glycine levels, and only oligomycin lowered coenzyme A levels in liver. However, the oligomycin-induced decline of hepatic coenzyme A levels was delayed, contrary to impairment of glycine conjugation, which was almost immediate. In summary, impairment of benzoylglycine formation by ATP depletors apparently correlates with their capacity to diminish ATP levels in hepatic mitochondria (i.e. at the site of glycine conjugation). These observations suggest that limited availability of mitochondrial (but not cytosolic) ATP reduces glycine conjugation capacity. Therefore, mitochondrium toxic agents and pathological mitochondrial injuries acting in liver may compromise glycine conjugation by decreasing ATP supply.

Lipoic acid impairs glycine conjugation of benzoic acid and renal excretion of benzoylglycine.

Glycine conjugation of benzoic acid is catalyzed by the mitochondrial enzymes benzoyl-coenzyme A(CoA) synthetase and benzoyl-CoA: glycine N-acyltransferase and requires ATP, CoA, and glycine as cosubstrates. Lipoic acid (LA), an important endogenous and also therapeutic compound, depletes hepatic CoA; therefore, it may interfere with glycine conjugation. To test this hypothesis, LA (0.5-1.5 mmol/kg ip) was given to anesthetized, glycine-loaded rats 1 hr before administration of benzoic acid (1 mmol/kg iv). LA inhibited glycine conjugation of benzoic acid in a dose-dependent manner as indicated by: 1) reduced clearance of benzoic acid from blood; 2) delayed appearance of benzoylglycine in blood; and 3) decreased excretion of benzoylglycine in urine. LA also decreased urinary excretion of injected benzoylglycine, indicating that reduced excretion of this metabolite after benzoic acid injection is caused by diminished formation and impaired renal transport of benzoylglycine. Urine formation was decreased by LA in a dose-dependent fashion, and acute renal failure was evident in rats receiving the highest dose. LA depleted hepatic CoA, carnitine, and glutathione, but not ATP, whereas it increased the hepatic concentration of glycine. In isolated and solubilized rat liver mitochondria, LA inhibited both benzoyl-CoA synthetase (IC50 approximately 1.5 mM) and benzoyl-CoA:glycine N-acyltransferase (IC50 approximately 0.3 mM). Thus, depletion of CoA and inhibition of the pertinent enzymes seem responsible for impairment of glycine conjugation of benzoic acid by LA. LA may also impair renal tubular cell function, compromising the tubular secretion of benzoylglycine and causing acute renal failure.

Simple method for analysis of diquat in biological fluids and tissues by high-performance liquid chromatography.

A simple HPLC method has been described to quantify diquat in biological fluids and tissues. This method permits separation and quantification of diquat from blood, bile, urine, liver and kidney. It does not require special pretreatment of the samples prior to analysis, nor a specially prepared analytical column. Various concentrations of diquat were added (10-300 nmol/ml or g) to fluids or tissues. Analysis of blank samples revealed no substances that interfere with diquat elution. Excellent recovery (95-105%) was obtained. Diquat (120 mumol/kg, i.v.) was injected to rats and quantified in bile, blood and liver. Concentration of diquat was higher in blood and bile than liver. Therefore, this method is applicable for quantification of diquat in toxicological samples, and may be used to determine structurally similar compounds such as paraquat.

Sulfation of acetaminophen and acetaminophen-induced alterations in sulfate and 3'-phosphoadenosine 5'-phosphosulfate homeostasis in rats with deficient dietary intake of sulfur.

Sulfation of drugs depends on the availability of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), which requires inorganic sulfate for its synthesis. Therefore, decreased alimentary intake of inorganic sulfate or its precursor, cysteine, may compromise sulfation of xenobiotics. To test this hypothesis, separate groups of rats were maintained for 5 days on synthetic diets, which lacked sulfate, or cysteine, or both sulfate and cysteine. These dietary restrictions did not cause growth retardation or depletion of glutathione in liver. Under anesthesia, the animals were injected with acetaminophen (0.5 mmol/kg, i.v.) and elimination of acetaminophen from blood and excretion of acetaminophen metabolites in urine and bile was simultaneously quantified. Deficient intake of inorganic sulfate or cysteine alone did not significantly change elimination and biotransformation of acetaminophen. Combined nutritional deficiency of sulfate and cysteine, however, resulted in a 40% reduction in the excretion of acetaminophen-sulfate, quantitatively the most significant metabolite. Concomitantly, these animals eliminated acetaminophen from blood at a slower rate and converted more acetaminophen to its toxic intermediate, as indicated by increased excretion of acetaminophen-thioether conjugates. Serum and tissue sulfate concentrations were decreased to significantly lower levels in rats on sulfate and cysteine deficient diets, than in rats with a sufficient sulfur supply. Thus, reduced sulfation is apparently caused by diminished availability of inorganic sulfate for PAPS synthesis, even though hepatic and renal PAPS levels were not depleted more by acetaminophen in rats with deficient dietary supply of sulfate and cysteine than in rats with adequate sulfur intake.(ABSTRACT TRUNCATED AT 250 WORDS)

Molybdate depletes hepatic 3-phosphoadenosine 5-phosphosulfate and impairs the sulfation of acetaminophen in rats.

Molybdate (15 mmol/kg p.o.) decreased serum sulfate concentrations of rats 70% within 6 hr after administration. Parallel to this depletion, there was a dramatic decrease in hepatic sulfate and 3-phosphoadenosine 5-phosphosulfate (PAPS) concentrations (about 40 and 65%, respectively). However, renal PAPS concentrations did not change significantly. Molybdate reduced serum, hepatic and renal sulfate as well as hepatic PAPS concentration in a dose-dependent manner up to the dose of 10 mmol/kg. However, renal PAPS did not change. The results indicate that molybdate reduced not only sulfate concentrations in serum and tissue, but also PAPS concentrations in liver. The effect of molybdate on the pharmacokinetics of acetaminophen (AA, 150 mg/kg i.v.) was also investigated in order to determine whether molybdate-induced depletion of PAPS might be a useful tool for examining the importance of sulfation in the detoxication and toxication of xenobiotics. AA-sulfate concentration in blood decreased 40 and 80% after administration of molybdate at doses of 2.5 and 15 mmol/kg, respectively. Molybdate also decreased the excretion of AA-sulfate into bile and urine by about 60 and 80%, respectively. However, molybdate did not alter the excretion of AA-glucuronide and AA-glutathione/cysteine. The excretion of the parent AA increased 2-fold after molybdate administration (15 mmol/kg). In conclusion, molybdate effectively lowers inorganic sulfate in serum and tissues, and PAPS in the liver. Reduction of hepatic PAPS markedly decreases the sulfation of AA, suggesting that molybdate treatment could be used to study the importance of sulfation in pharmacology and toxicology.

Nutritionally and chemically induced impairment of sulfate activation and sulfation of xenobiotics in vivo.

Sulfation requires 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as the sulfate donor. In the search for methods to inhibit sulfation reactions via impairment of PAPS synthesis, two experimental conditions have been tested in rats. A low-sulfur diet, which does not deplete hepatic glutathione, reduced inorganic sulfate but not PAPS levels in the liver and moderately decreased sulfation of acetaminophen. Administration of molybdate, which is an alternative substrate for intestinal and renal sulfate transport as well as for ATP-sulfurylase, depleted both sulfate and PAPS in liver and markedly inhibited sulfation of acetaminophen. Therefore, administration of molybdate may be used as an experimental tool to study the role of sulfation in the fate and effect of xenobiotics.

Effect of valproic acid on glycine conjugation of benzoic acid.

Conjugation with glycine proceeds through ATP-dependent coupling of carboxylic acids with coenzyme A (CoA). Therefore, chemicals that form CoA esters may interfere with glycine conjugation. We tested the hypothesis that valproic acid (VPA), which is esterified with CoA in the first step of its mitochondrial beta-oxidation, may compromise glycine conjugation of aromatic carboxylic acids, by investigating the effect of acute VPA administration on glycine conjugation of benzoic acid in rats. VPA administered 1 hr before injection of benzoate only decreased the blood clearance of benzoate and the urinary excretion of benzoylglycine slightly in normal rats. However, in rats loaded with glycine, 2 and 3 mmol/kg of VPA reduced the blood clearance of benzoate by 34 and 59%, diminished the peak blood level of the glycine conjugate and depressed the maximal urinary excretion rate of benzoylglycine by 28 and 66%, respectively. To elucidate the mechanism of VPA-induced inhibition of benzoylglycine formation, the effects of VPA on hepatic levels of cosubstrates and the activities of enzymes involved in glycine conjugation were also determined. One hour after administration of VPA, hepatic ATP levels remained unchanged, whereas the concentration of CoA was reduced by 67 to 73% and that of glycine was increased by 58 to 67%. Activities of the enzymes of glycine conjugation were not influenced by VPA. However, 2-n-propyl-4-pentenoic acid, a metabolite of VPA, inhibited benzoyl-CoA synthetase. In summary, VPA minimally influenced the capacity of glycine conjugation of benzoic acid in normal rats, but decreased it markedly in glycine-loaded rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Dependence of glycine conjugation on availability of glycine: role of the glycine cleavage system.

1. Glycine conjugation of benzoic acid was investigated in anaesthetized rats by measuring the disappearance of benzoate from blood, and the appearance of benzoylglycine in blood and urine. 2. Administration of glycine (1-10 mmol/kg,i.v.) increased the capacity of benzoylglycine formation in a dose-dependent fashion, with a maximal rate (8.1 mumol/kg per min) occurring after administration of 5 mmol/kg glycine. The normal endogenous glycine supply (1.7 mM in liver) permits glycine conjugation only at an approximate half-maximal rate (4.5 mumol/kg/per min). 3. The increase in benzoylglycine formation in response to exogenous glycine supply is also a function of the benzoate dosage. Decreased responsiveness at high benzoate dosage indicates that the availability of coenzyme A is another factor that also limits the capacity of glycine conjugation. 4. Cysteamine (200 mg/kg, i.p.), a potent inhibitor of the mitochondrial glycine cleavage system, rapidly increased hepatic glycine concentration 2-3-fold without affecting the concentration of the other co-substrates (i.e. coenzyme A and ATP) of glycine conjugation. 5. Administration of cysteamine increased the blood clearance of benzoate by 50%, the appearance of benzoylglycine in blood, and the urinary excretion of benzoylglycine. 6. It is concluded that the activity of glycine cleavage system is an important determinant of glycine supply and, thereby, the capacity of glycine conjugation of xenobiotics.

Marked interanimal differences in susceptibility of Sprague-Dawley rats to diquat-induced oxidative stress in the liver: correlation with hepatic uptake of diquat.

Biliary excretion of oxidized glutathione (GSSG) is used as an index of oxidative stress. We observed a marked interanimal difference in susceptibility to diquat-induced oxidative stress. When diquat injections (120 mumol/kg, i.v.) were administered to rats, a 60-fold increase in the biliary excretion of GSSG was observed in 40% of the rats (responders); however, diquat failed to increase the biliary excretion of GSSG in 60% of the animals (nonresponders). This interanimal variation is not due to differences in the hepatic metabolism or hepatobiliary transport of GSSG, as no interanimal difference was observed in the biliary output of GSSG after administration of another oxidative stress-inducing agent, t-butyl hydroperoxide (1.4 mmol/kg, i.v.). We then examined the hepatobiliary disposition of diquat (120 mumol/kg, i.v.) using a high-performance liquid chromatography procedure to quantitate diquat in blood, liver and bile. No differences in blood or biliary concentration of diquat were noted between responders and nonresponders. However, a marked difference was observed in the hepatic concentration of diquat in responders and nonresponders. The responders exhibited a 4-fold higher hepatic diquat concentration than the nonresponders (65 or 15 nmol/g, respectively) 30 min after diquat administration. In conclusion, this study demonstrates a marked interanimal variation in the susceptibility of Sprague-Dawley rats to oxidative stress produced by diquat, which appears to be due to interanimal difference in the hepatic accumulation of diquat.