Formation of a diazonium cation intermediate in the metabolism of sulphamethazine to desaminosulphamethazine in the rat

14C-Sulphamethazinediazonium tetrafluoroborate (14C-SDTFB) when orally administered to rats was converted primarily to 14C-labelled desaminosulphamethazine (desaminosulmet) and methanol-insoluble residues in the gastrointestinal tract (gut). 14C-labelled sulphamethazine (sulmet), N4-acetylsulmet, the N4-glucose conjugate of sulmet and other unidentified products were also observed in the tissues and urine of rats given 14C-SDTFB. 2. When 14C-sulmet, nitrite and dimethylaniline were simultaneously administered to a rat by the oral route, one of the 14C-labelled products formed in the stomach was isolated and identified as 4-dimethylaminophenyl [4-(N-4,6-dimethyl-2-pyrimidinyl)sulphamidophenyl] diazene, providing evidence that 14C-sulmet was diazotized in the stomach of the animal. 3. SDTFB was weakly mutagenic when evaluated by the Ames test. 4. The methanol-insoluble 14C-labelled residues in the gut of rats dosed orally with 14C-SDTFB and 14C-sulmet + nitrite were partially converted to 14C-labelled desaminosulmet, sulmet, N4-acetylsulmet and other unidentified products when fed to recipient rats.     

The effect of dietary nitrite and nitrate on the metabolism of sulphamethazine in the rat

Rats given 100 p.p.m. of 14C-sulphamethazine [4-amino-N-(4,6-dimethyl-2-pyrimidinyl)benzene[U-14C]sulphonamide] in the diet excreted less 14C-activity in the urine as the amount of nitrite (0 to 1000 p.p.m.), but not nitrate (3730 p.p.m.), in the diet was increased. As the level of nitrite, but not nitrate, was increased, there was a concomitant increase in the amount of 14C-desaminosulphamethazine (4-[(N-4,6-dimethyl-2-pyrimidinyl)benzene-[U-14C]-sulphonamide in the blood, liver, skeletal muscle and gastrointestinal tract. As the level of nitrite, but not nitrate, supplementation was increased, the amount of methanol-insoluble 14C-activity in the gastrointestinal tract increased but the amount of insoluble 14C-activity in the blood, skeletal muscle and liver was not changed.     

Identification of intermediate pathways of 4-hydroxynonenal metabolism in the rat

The formation of 4-hydroxy-2-nonenal (HNE) conjugates with glutathione (GSH) by Michael addition and subsequent cleavage to yield the related mercapturic acid (MA) conjugates are a major detoxication process. To characterize the metabolic pathways involved in the formation of urinary HNE-MA conjugates in the rat, the metabolism of HNE-thioethers (HNE-GSH, HNE-MA, and HNE-Cys) by rat liver and kidney cytosolic fractions was investigated. The experimental results showed that HNE-GSH is a good substrate for cytosolic incubations whereas HNE-MA and HNE-Cys are poorly metabolized. About 80% of the urinary MA conjugates originate from the primary and major HNE metabolite, namely, the hemiacetalized HNE-GSH. The direct reduction of HNE-GSH by a cytosolic aldo-keto reductase (NADPH) leads to 1,4-dihydroxynonene-GSH (DHN-GSH) and subsequently to DHN-MA. The direct oxidation of HNE-GSH by aldehyde dehydrogenase (NAD)(+) leads to 4-hydroxynonenoic-lactone-GSH, the partial hydrolysis of which occurs at physiological pH and accounts for the corresponding 4-hydroxynonenoic-GSH. Both the spontaneous- and the glutathione S-transferases-catalyzed retro-Michael cleavages of HNE-GSH and HNA-lactone-GSH are the source of HNE and HNA-lactone, respectively. This latter compound, with both lipophilic and electrophilic properties, is available for microsomal omega-hydroxylation by cytochrome P450 4A enzymes and conjugation with thiol groups and therefore is the most likely candidate for the formation of omega-hydroxylated HNE-mercapturic acid conjugates excreted in rat urine.     

The metabolism and deamination of [14C]sulphamethazine in a germ-free pig: the influence of nitrate and nitrite

Twenty-four hours after feeding nitrite in combination with [14C]sulphamethazine to a germ-free and a conventional control pig the level of [14C]desaminosulphamethazine in tissues from both pigs was high, accounting for 11 to 30% of the total tissue 14C. When another germ-free pig was fed [14C]sulphamethazine in combination with nitrate, a trace amount of [14C]desaminosulphamethazine was found by gas chromatography-mass spectroscopy in the skeletal muscle but not in other tissues. When a control pig was fed [14C]sulphamethazine and nitrate, [14C]desaminosulphamethazine was found in all tissues examined. The results from this study show that feeding pigs nitrite together with sulphamethazine increases the amount of desaminosulphamethazine in the tissues. Most of the desaminosulphamethazine found in the tissues of the control pig fed nitrate was presumably the secondary effect of bacterial reduction of nitrate to nitrite.     

Dehydro-digitoxosides of digitoxigenin: Formation and importance for the digitoxin metabolism in the rat

Summary Only in the presence of NADPH and oxygen digitoxosides of digitoxigenin can be metabolized by rat liver microsomes. The metabolism includes 12-hydroxylation, formation of digitoxosides less the terminal digitoxose and of lipophilic metabolites. The lipophilic metabolites of digitoxin (dt-3), digitoxigeninbis-digitoxoside (dt-2), and of — mono-digitoxoside (dt-1) are formed by oxidation of the axial OH-group of the terminal digitoxosyl yielding the corresponding dehydro-digitoxosides. The structure could be confirmed by comparison with the synthetic compounds 15-dehydro-dt-3, 9-dehydro-dt-2, and 3-dehydro-dt-1, respectively.The terminal dehydro-digitoxosyl can be split off by liver microsomes even in the absence of NADPH. However, no further cleavage of the resulting digitoxosides could be detected. A cleavage rate of 2.8 nmoles/mg microsomal protein × 30 min was observed for both 15-dehydro-dt-3 and 9-dehydro-dt-2 (substrate concentration 50 M). In contrast to that, only 0.4 nmole 3-dehydro-dt-1 was cleaved under equal conditions. For the cleavage of 15-dehydro-dt-3 an apparent K _m of 200 M and a V _max of 440 pmoles dt-2 formed per mg microsomal protein x min was measured.Our results indicate that not the native but only the dehydro-digitoxosides can be cleaved. Therefore, two successive monoxygenase catalysed oxidations are necessary for the cleavage of the sugar chain of dt-3 before dt-1, the main substrate of conjugation enzymes, can be formed. Moreover, digitoxigenin will not be formed because of the high stability of 3-dehydro-dt-1.     

The role of oxidative and glycolytic metabolism in cation transport of the isolated rat liver perfused with an erythrocyte-free medium

Summary Rat livers perfused with a medium devoid of red cells do not release remarkable amounts of potassium when O₂ is substituted by N₂ for 30 min, nor does 5×10^–5 M 2,4 DNP much increase the K⁺ concentration in the perfusate. On the other hand, high concentrations of sodium fluoride (7×10^–3 M) produce a pronounced K⁺ efflux and Na⁺ uptake. These findings suggest that a) the cation equilibirium is relatively insensitivite to transient O₂ deficiency and b) the active alkali cation transport may be coupled to the glycolytic energy supply in the liver.This work was supported by the Deutsche Forschungsgemeinschaft.     

In vitro metabolism of a model cyclopropylamine to reactive intermediate: insights into trovafloxacin-induced hepatotoxicity

Trovafloxacin (Trovan) is a fluoroquinolone antibiotic drug with a long half-life and broad-spectrum activity. Since its entry into the market in 1998, trovafloxacin has been associated with numerous cases of hepatotoxicity, which has limited its clinical usefulness. Trovafloxacin possesses two substructural elements that have the potential to generate reactive intermediates: a cyclopropylamine moiety and a difluoroanilino system. The results presented here describe the in vitro metabolic activation of a synthetic drug model (DM) of trovafloxacin that contains the cyclopropylamine moiety. Cyclopropylamine can be oxidized to reactive ring-opened products-a carbon-centered radical and a subsequently oxidized alpha,beta-unsaturated aldehyde. Experiments with monoamine oxygenases, horseradish peroxidase, flavin monooxygenase 3, and cDNA-expressed P450 isoenzymes revealed that P450 1A2 oxidizes DM to a reactive alpha,beta-unsaturated aldehyde, M 1. Furthermore, myeloperoxidase (MPO) was also demonstrated to oxidize DM in the presence of chloride ion to produce M 1. DM proved to be a suicide inhibitor of MPO while showing no inhibition of P450 1A2. The structure of the reactive metabolite was confirmed by LC-MS/MS analysis by comparison with a synthetic standard. M 1 was further shown to react with glutathione and the related thiol nucleophile, 4-bromobenzyl mercaptan, suggesting the potential of this intermediate to react with protein nucleophiles. In summary, these data provide evidence that trovafloxacin-induced hepatotoxicity may be mediated through the oxidation of the cyclopropylamine substructure to reactive intermediates that may form covalent adducts to hepatic proteins, resulting in damage to liver tissue.     

Pharmacokinetics of pinokalant, a new nonselective cation channel blocker in the rat.

The pharmacokinetics of 1-isoquinolineacetamide, 3,4-dihydro-6,7-dimethoxy-alpha-phenyl-N,N-bis [2-(2,3,4-trimethoxyphenyl)ethyl]-, monomethanesulfonate (pinokalant, salt form of the active entity LOE 908 BS, CAS 143482-63-7), a nonselective cation channel blocker, was studied in rats. Drug plasma levels declined rapidly in a polyphasic manner after intravenous bolus administration of 8.8 mg/kg LOE 908 BS. The disposition of LOE 908 BS was governed by a rapid elimination (clearance Cl = 47.7 ml/min/kg) and an extensive distribution into tissues (volume of distribution Vss = 7.21 l/kg). A dose-proportional increase of AUC and steady state concentration up to doses of 194 mg/kg (6 h infusion) was observed suggesting linear pharmacokinetics. The protein binding was very high with 99.4% to 99.7% bound to plasma proteins in the concentration range 0.26 to 2.6 micrograms/ml. The LOE 908 BS concentration-time profile in brain tissue after intravenous infusion (4.4 mg/kg/h over 4 h) paralleled those measured in plasma indicating a rapid but also low penetration of the blood-brain-barrier. The concentration-time profile of drug-related radioactivity after intravenous (bolus) administration of [14C]LOE 908 BS dropped also rapidly to approximately 16% within the first hour compared to the initial 2-min value. The drug exhibited a high biliary excretion (84% during 5 h) and, accordingly, faecal excretion was the main route of excretion (> 90%). The mass balance was complete after 96 h indicating no persistence of radioactivity in the animals. The relevance of these findings with respect to results obtained with LOE 908 BS in animal models for stroke and traumatic brain injury is discussed.     

Palytoxin induces a nonselective cation channel in single ventricular cells of rat

Summary Palytoxin (PTX) causes a depolarization in every excitable tissue tested. We examined whether PTX induces a channel current, which might be responsible for the depolarizing action, in single ventricular cells from rat hearts using a patch-clamp technique. In cell-attached configuration, when PTX (30 pmol/1) was present in a patch pipette, a single channel current appeared, which was different from currents through known channels. When the major cation in the pipette was Na⁺, the amplitude of the unitary current at the resting potential level was 0.74 ± 0.05 pA, the slope conductance was 9 pS and the reversal potential was 79 mV positive to the resting potential. The channel was little selective between cations because it was permeable not only to Na⁺ but also to K⁺, Cs⁺ and Li⁺ although it practically did not permit the passage of choline+, TEA⁺, Bat⁺ and Ca²⁺. Thus PTX induces a novel, nonselective cation channel in cardiac cells, which may result in a depolarization. Send offprint requests to K. Ito     

Diethylstilbestrol (DES) quinone: a reactive intermediate in DES metabolism

The quinone of E-diethylstilbestrol (DES), a postulated metabolic intermediate derived from DES, has been synthesized by oxidation of DES in chloroform using silver oxide. The reaction product was structurally characterized by infrared, ultraviolet, nuclear magnetic resonance, and mass spectrometry. The product of oxidation of DES by hydrogen peroxide, catalyzed by horseradish peroxidase and also by rat uterine peroxidase, was shown to be identical with synthetic DES quinone based on identical u.v. spectra and on identical decomposition products. DES quinone was stable only in non-protic solvents such as chloroform. In acids, bases or protic solvents, DES quinone rearranged to Z,Z-dienestrol (beta-DIES). The half-life of DES quinone in water was approximately 40 min; in methanol it was approximately 70 min. Bacterial mutagenicity (Ames) tests did not indicate that DES quinone had mutagenic or genotoxic activity. However, DES quinone was found to bind to calf thymus DNA without any enzyme mediation at levels significantly above the binding of DES under the same conditions. Based on the binding of DES quinone to DNA, this intermediate must be considered as a possible carcinogenic metabolite of DES.     

Metabolism of the dyestuff intermediate 2,4-diaminoanisole in the rat

1. 2,4-Diamino[ring-U-¹⁴C] administered intraperitoneally to rats is excreted chiefly via the urine (79 and 85% of the dose in 24 and 48h, respectively). The isotope in the faeces was 2·1 and 8·9% of the dose at 24 and 48 h.

2. The major metabolic pathway was acetylation of the amine group(s), resulting in 4-acetylamino-2-aminoanisole and 2,4-diacetylaminoanisole.

3. Oxidative pathways yielded 2,4-diacetylaminophenol (O-demethylation), 5-hydroxy-2,4-diacetylaminoanisole (ring hydroxylation), and 2-methoxy-5-(glycolamido)acetanilide or its isomer (ω-oxidation).

4. These major metabolites were excreted in the urine both as free and glucuronic acid conjugates.

1. A single oral dose of desmethylimipramine (80mg/kg) administered to rats inhibited the hepatic microsomal hydroxylation of thiabendazole (45%), aniline (30%), biphenyl (30%) and ethylmorphine (15%) in vitro at 5 h after dosage; there was no decrease in cytochrome P-450 or b₅.

2. A single oral dose of ethoxyquin (200mg/kg) to rats inhibited the hepatic microsomal hydroxylation of thiabendazole (65%), aniline (40%) and biphenyl (40%) in vitro at 1 h after dosage; inhibition was less at 5 h. There were no changes in the contents of cytochromes P-450 and b₅.

3. The max. plasma concn. of thiabendazole occurred 2-4h after oral dosing (50-200mg/kg) to rats. Thiabendazole (100mg/kg) administered orally 30min after oral ethoxyquin (400 mg/kg) or thiabendazole (200 mg/kg) administered orally 30 min after oral desmethylimipramine (80 mg/kg) delayed absorption of the thiabendazole and resulted in markedly decreased plasma concentration of the anthelmintic.

4. Simultaneous administration of ethoxyquin (300 mg/kg) potentiated the anthelmintic effect of thiabendazole (750 mg/kg) on the helminth parasite, Nematospiroides dubius, the mouse. Desmethylimipramine showed no similar potentiation.

1. The α-isomer of 1,2,3,4,5,6-hexachlorocyclohexane (HCH) is converted, on incubation with the 100 000g supernatant fraction (cytosol) of rat liver homogenates, to four positional isomers of S-(dichlorophenyl)glutathione (DCPG).

2. Radiochemical evidence shows that primary conjugates formed are subject to rapid aromatization.

3. The GSH-conjugates produced from γ-HCH (lindane), Δ-HCH, and three stereoisomers of 1,3,4,5,6-pentachlorocyclohex-1-ene (PCCH) have been characterized by g.l.c. of their aromatic moieties. All compounds were exclusively converted to at least two positional isomers of DCPG. An isomer of HCH and its trans-dehydrochlorination product yielded DCPG of almost identical composition.

4. β-HCH was not entirely unreactive, but the identity of the product remained uncertain.

5. DCPG-formation from HCH-d₆ exhibited a significant deuterium isotope effect (5·8 at 310 K for the α-configuration), while none was found for the conversion of PCCH-d₅ (1·2 at 298 K for the 3,4,6/5-isomer).

6. In the absence of GSH, liver cytosol protein mediated a trans-dehydrochlorination of lindane and of α-HCH to PCCH with a catalytic factor of 15 and 25, respectively. Addition of GSH raised HCH conversion by a factor of 3 to 4 and resulted in the formation of DCPG.

7. GSH-conjugation of PCCH is shown to be enzymic.

8. It is concluded that the rate of formation of DCPG from HCH in rat liver cytosol depends on gradual monodehydrochlorination, and that the enzymic transfer of GSH onto PCCH is not preceded by a second dehydrochlorination. The transfer and elimination reactions involved in DCPG formation from PCCH are considered taking into account (a) differences between stereoisomers in reaction rate and product composition and (b) the observation that a purified soluble GSH-S-transferase (E.C. 2.5.1.18) converted 3,4,6/5-PCCH-and α-HCH-to the same set of four isomeric DCPGs as did the entire cytosol fraction.

9. In corroboration of earlier evidence, transferase activity associated with liver microsomes and mitochondria also converts α-HCH and 3,4,6/5-PCCH each to four positional isomers of DCPG.

10. The result of the study is discussed with reference to earlier work in mammals and in insects and in relation to HCH-biotransformation by rats in vivo.

1. The disposition of spirohydantoin mustard (SHM) has been examined in rats and dogs after i.v. administration of [hydantoin-4-¹⁴C] SHM and [2-chloroethyl-U-¹⁴C] SHM.

2. Four hours after dosing to rats and dogs, renal clearance of the ethyl-¹⁴C moiety (14-21%) is lower than that of the hydantoin-¹⁴C moiety (29-35%). In contrast, biliary excretion of the ethyl-¹⁴C in rats is greater, and there appears to be enterohepatic circulation of the ethyl-¹⁴C.

3. Less than 1% of the radioactivity appearing in rat bile is unchanged SHM. Two major metabolites containing the ethyl-¹⁴C moiety are conjugates of glutathione or cysteine.

4. Levels of ¹⁴C in plasma decline in a biphasic manner. No difference in the initial plasma disappearance of the two labelled moieties is observed, but disappearance of the ethyl-¹⁴C during the second phase is slower.

5. Concentrations of ethyl-¹⁴C and hydantoin-¹⁴C in the brains of rats and dogs, 4h after i.v. administration, are equivalent to those in plasma.

6. Radioactivity is also distributed to the brain of rats after oral administration of [2-chloroethyl-U-¹⁴C]SHM, but at a concn. less than half that of plasma.     

Studies on the metabolism of tolmetin to the chemically reactive acyl-coenzyme A thioester intermediate in rats.

Carboxylic acids may be metabolized to acyl glucuronides and acyl-coenzyme A thioesters (acyl-CoAs), which are reactive metabolites capable of reacting with proteins in vivo. In this study, the metabolic activation of tolmetin (Tol) to reactive metabolites and the subsequent formation of Tol-protein adducts in the liver were studied in rats. Two hours after dose administration (100 mg/kg i.p.), tolmetin acyl-CoA (Tol-CoA) was identified by liquid chromatography-tandem mass spectrometry in liver homogenates. Similarly, the acyl-CoA-dependent metabolites tolmetin-taurine conjugate (Tol-Tau) and tolmetin-acyl carnitine ester (Tol-Car) were identified in rat livers. In a rat bile study (100 mg/kg i.p.), the S-acyl glutathione thioester conjugate was identified, providing further evidence of the formation of reactive metabolites such as Tol-CoA or Tol-acyl glucuronide (Tol-O-G), capable of acylating nucleophilic functional groups. Three rats were treated with clofibric acid (150 mg/kg/day i.p. for 7 days) before dose administration of Tol. This resulted in an increase in covalent binding to liver proteins from 0.9 nmol/g liver in control rats to 4.2 nmol/g liver in clofibric acid-treated rats. Similarly, levels of Tol-CoA increased from 0.6 nmol/g to 4.4 nmol/g liver after pretreatment with clofibric acid, whereas the formation of Tol-O-G and Tol-Tau was unaffected by clofibric acid treatment. However, Tol-Car levels increased from 0.08 to 0.64 nmol/g after clofibric acid treatment. Collectively, these results confirm that Tol-CoA is formed in vivo in the rat and that this metabolite can have important consequences in terms of covalent binding to liver proteins.     

Formaldehyde metabolism by the rat: a re-appraisal

1. The metabolism of [14C]formaldehyde has been investigated in the male Sprague-Dawley rat. It is extensively oxidized to CO2 and formate, which is excreted in the urine. 2. Two radioactive compounds isolated from the urine of rats dosed with [14C]formaldehyde have been identified as N-hydroxymethylurea and N,N'-bis-(hydroxymethyl)urea, and shown to be urinary artefacts. 3. Previous studies of the metabolism of formaldehyde by rats have been re-appraised. Differences in the rate of oxidation of formaldehyde in various strains of rats result in the excretion of different urinary metabolites and, in some cases, formaldehyde. Excretion of formaldehyde leads to the formation of several artefacts depending on the components present in the urine.     

Vesicular uptake system for the cation lucigenin in the rat hepatocyte

The hepatic transport mechanism for the fluorescent bivalent hydrophilic organic cation lucigenin (LU) was characterized employing kinetic and morphological methods. The extraction of LU by the perfused rat liver was 50% and uptake was saturable. LU did not inhibit the carrier-mediated hepatic uptake of the model organic cationic compounds tributylmethyl ammonium (type 1) and vecuronium (type 2) in isolated hepatocytes, whereas the uptake of LU in the perfused liver was not affected by either type of cation or by the cardiac glycoside cymarin, a potent type 2 inhibitor. The cytoskeleton-disrupting agents cytochalasin B and nocodazole, however, significantly lowered hepatic uptake of LU. In the intact liver, LU did not stimulate fluid phase endocytosis, as indicated by a lack of effect on the internalization of horseradish peroxidase. These kinetic data point to adsorptive endocytosis as the most probable uptake mechanism. This was confirmed by the inhibitory effect of neomycin and the polycation poly(L-lysine) on LU uptake. Fluorescence microscopy revealed that LU accumulated in the hepatocytes in discrete vesicular structures. Partial co-localization of rhodamine-dextran and acid phosphatase with LU indicated that part of the LU fluorescence was present in lysosomes, although not all lysosomes contained LU. Taken together, we conclude that we identified a novel vesicular pathway for uptake of organic cations by hepatocytes.     

Metabolism of the dyestuff intermediate 2,4-diaminoanisole in the rat.

1. 2,4-Diamino[ring-U-14C]anisole.2HCl administered intraperitoneally to rats is excreted chiefly via the urine (79 and 85% of the dose in 24 and 48 h, respectively). The isotope in the faeces was 2.1 and 8.9% of the dose at 24 and 48 h. 2. The major metabolic pathway was acetylation of the amine groups(s), resulting in 4-acetylamino-2-aminoanisole and 2,4-diacetylaminoanisole. 3. Oxidate pathways yielded 2,4-diacetylaminophenol (O-demethylation), 5-hydroxy-2,4-diacetylaminoanisole (ring hydroxylation), and 2-methoxy-5-(glycol-amido)acetanilide or its isomer (omega-oxidation). 4. These major metabolites were excreted in the urine both as free and glucuronic acid conjugates.