Multidrug resistance protein 2-mediated estradiol-17beta-D-glucuronide transport potentiation: in vitro-in vivo correlation and species specificity.

Multidrug resistance protein 2 (MRP2) is a multispecific organic anion transporter expressed at important pharmacological barriers, including the canalicular membrane of hepatocytes. At this location it is involved in the elimination of both endogenous and exogenous waste products, mostly as conjugates, to the bile. Estradiol-17beta-d-glucuronide (E(2)17betaG), a widely studied endogenous substrate of MRP2, was shown earlier to recognize two binding sites of the transporter in vesicular transport assays. MRP2 modulators (substrates and nonsubstrates) potentiate the transport of E(2)17betaG by MRP2. We correlated data obtained from studies of different complexities and investigated the species-specific differences between rat and human MRP2-mediated transport. We used vesicular transport assays, sandwich-cultured primary hepatocytes, and in vivo biliary efflux in rats. Our results demonstrate that the rat Mrp2 transporter, unlike the human MRP2, transports E(2)17betaG according to Michaelis-Menten type kinetics. Nevertheless, in the presence of modulator drugs E(2)17betaG transport mediated by the rat transporter also shows cooperative kinetics as potentiation of E(2)17betaG transport was observed in the vesicular transport assay. We also demonstrated that the potentiation exists both in rat and in human hepatocytes and in vivo in rats.

Staphylococci isolated from animals and food with phenotypically reduced susceptibility to beta-lactamase-resistant beta-lactam antibiotics.

The antibiotic resistance pattern of 1921 Staphylococcus strains isolated from animals and food within the last two years were examined using diffusion tests. Among them there were only 35 strains of S. aureus having an inhibition zone diameter of 15 mm or less, and 4 strains of coagulase-negative staphylococci (CNS) having a zone diameter of 18 mm or less to 1-microg oxacillin disk. These 39 strains were examined also by E-test to oxacillin and for the detection of the mecA gene by PCR in order to determine whether they might be real methicillin-resistant staphylococci. Among the 39 strains there were only two that were susceptible to penicillin by disk diffusion method; however, further examination by the penicillinase test showed that they produced beta-lactamase. While 19 (15 S. aureus, 4 CNS) strains were resistant and 7 strains were intermediate to oxacillin in disk diffusion test, the E-test gave 8 resistant and 5 intermediate results. Six out of the 8 oxacillin-resistant strains examined by disk diffusion and E-test harboured the mecA gene. Thus only 6 out of the examined 1921 strains proved to be mecA positive. These methicillin-resistant, mecA-positive strains (5 of the S. aureus strains and 1 of the S. epidermidis) originated from two dairy herds. The results prove that methicillin-resistant S. aureus (MRSA) strains in animals are really rare in Hungary. Eighteen strains were chosen and screened for minimal inhibitory concentration (MIC) of oxacillin with or without clavulanic acid or sulbactam, and three of them produced methicillinase enzyme.

Effect of alpha-methyldopa on pentoxyresorufin O-dealkylation in liver microsomes from rats treated with phenobarbital.

Loss of pentoxyresorufin O-dealkylation (PROD) was observed when microsomes from PB-treated rats were preincubated in the presence of NADPH. PROD proved to be quite sensitive towards inactivation. Decrease in cytochrome P450 (CYP) dependent activity was accompanied by simultaneous formation of thiobarbituric acid reactive substances (TBARS) indicating the occurrence of lipid peroxidation. The presence of 50 microM alpha-methyldopa (AMD) during preincubation with NADPH resulted in complete protection against enzyme activity loss and the extent of lipid peroxidation was also diminished. Addition of ascorbate or GSH in combination with AMD reduced the protective effect of the drug on PROD. AMD probably exerts its effect by scavenging reactive oxygen species but chelation of ferric ions can also contribute to the protective effect of the drug on PROD activity.

Effect of phenobarbital and spironolactone treatment on the oxidative metabolism of antipyrine by rat liver microsomes.

The effects of pretreating rats with the inducers, phenobarbital or spironolactone, on the formation rate of the three major oxidative metabolites of antipyrine in vitro by hepatic microsomal fractions have been investigated. Both inducers reduced the rate of 3-methylhydroxylation of antipyrine by approximately 50%. In contrast, N-demethylation and 4-hydroxylation were enhanced 1.7-fold and 3.4-fold, respectively, in case of phenobarbital induction and 1.4-fold and 2.6-fold, respectively, following spironolactone treatment. To elucidate the role of some cytochrome P450 isoenzymes in the production of the three major metabolites of antipyrine, the effects of form selective enzyme inhibitors on antipyrine oxidation were also studied. Troleandomycin did not alter 3-methylhydroxylation but reduced both N-demethylation and 4-hydroxylation of antipyrine in microsomes from induced rat liver. Cimetidine and chloramphenicol decreased the rate of formation of all three metabolites in microsomes from induced and uninduced animal livers as well. Chloramphenicol seemed to be the most potent inhibitor of in vitro antipyrine oxidation. Alpha-methyldopa significantly enhanced the rate of formation of 4-hydroxyantipyrine and slightly reduced the rate of N-demethylation and 3-methylhydroxylation. According to the data obtained with microsomes from uninduced rat livers, the formation of the three major metabolites of antipyrine is extensively mediated by CYP2C11/C6. In microsomes from induced animal liver, CYP2B and CYP3A may contribute to both N-demethylation and 4-hydroxylation of antipyrine.

In vitro-in vivo correlation of the pharmacokinetics of vinpocetine.

Vinpocetine is extensively metabolized in rats, dogs and humans, and the plasma clearance approximates the hepatic plasma flow in each of the species. In vitro degradation studies with hepatocytes have shown that the activity of human hepatocytes is about one order of magnitude higher than the activity of dog hepatocytes, and two orders of magnitude higher than that of rat hepatocytes. These differences can explain the differences in bioavailabilities of vinpocetine in the three species (52% in rats, 21.5+/-19.3% in dogs and 6.2+/-1.9% in humans). In dogs and humans, the compound seems to be metabolized exclusively in the liver whereas in rats extrahepatic metabolism seems also to be important. The in vivo clearance predicted from the activity of hepatocytes is in good agreement with the values measured in vivo in the case of humans and dogs. The estimated values for bioavailability showed good correlation with in vivo data in each species if the free drug ratio was assumed to equal 1.

Glucuronidation of thyroxine in primary monolayer cultures of rat hepatocytes: in vitro induction of UDP-glucuronosyltranferases by methylcholanthrene, clofibrate, and dexamethasone alone and in combination.

Induction of UDP-glucuronosyltransferases (UGTs) toward thyroxine (T4) and p-nitrophenol (pNP) by 3-methylcholanthrene (MC), dexamethasone (DEX), clofibrate (Cl), and MC combined with DEX or Cl was studied in rat hepatocyte culture. We have developed a sensitive method for the measurement of glucuronide conjugates of the two substrates based on HPLC analysis of culture medium. MC, Cl, or DEX increased the activity of T4 UGT. Combination of MC and Cl showed additive effect, enzyme activity was enhanced compared with either MC or Cl treatment alone (617, 441, and 217% of the control, respectively). Combination of MC and DEX did not result in higher T4 UGT activity than MC treatment alone. Both MC and DEX enhanced the pNP UGT activity (182 and 162% of the control, respectively). Combination of MC with DEX resulted in additive effect. Cl treatment did not affect pNP conjugation either alone or in combination with MC. Western blot analysis revealed that only the amount of UGT1A1 was elevated by Cl and DEX. In contrast, concentration of UGT1A6 was increased by MC. Previous studies demonstrated that UGT1A1 inducers like phenobarbital have no effect on T4 conjugation (). Our results suggest that Cl, a known inducer of UGT1A1, enhances the activity of other enzyme(s) involved in T4 glucuronidation as well. It is well documented that DEX potentiates the inductory effect of polycyclic aromatic hydrocarbon on UGT1A6 (). In our study, MC increased the rate of T4 glucuronidation, and DEX had no additional effect on this reaction, suggesting that UGT1A6 is not the only enzyme inducible by MC that can catalyze T4 conjugation.

[Human drug metabolizing enzymes. I. Oxidative enzymes]. / Human gyógyszermetabolizáló enzimek. I. Oxidációs enzimek.

In this review we focus on human oxidative enzymes that are responsible for the metabolism of xenobiotics. 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.

A purified selenophosphate-dependent enzyme from Salmonella typhimurium catalyzes the replacement of sulfur in 2-thiouridine residues in tRNAs with selenium.

A tRNA-modifying enzyme tentatively termed tRNA 2-selenouridine synthase was purified by a five-step procedure that resulted in 50-60% pure preparations. This enzyme catalyzes the conversion of a 5-methylaminomethyl-2-thiouridine residue in the tRNA substrate to 5-methylaminomethyl-2-selenouridine. The selenium donor substrate for this reaction is shown to be selenophosphate which is formed from ATP and selenide by selenophosphate synthetase. Replacement of sulfur with selenium in tRNAs catalyzed by tRNA 2-selenouridine synthase occurs in the absence of ATP. The dependence of reaction velocity on selenophosphate concentration obeys Michaelis-Menten kinetics indicating an apparent Km value of 17.1 microM. Bulk thio-tRNA preparations from Escherichia coli and Salmonella typhimurium are equally effective as substrates for the selenium incorporation reaction. An intact 3' end of the tRNA molecule does not seem to be essential for selenium incorporation. Identity of the product of the reaction was confirmed by HPLC analysis of digests of [75Se]seleno-tRNAs labeled by incubation with the purified enzyme. A labeled compound in the nucleoside mixture was coeluted with authentic 5-methylaminomethyl-2-selenouridine.

Selenophosphate synthetase. Enzyme properties and catalytic reaction.

Selenophosphate synthetase, the product of the selD gene, produces the biologically active selenium donor compound, monoselenophosphate, from ATP and selenide. Isolation of the enzyme and characterization of some of its physical and catalytic properties are described. Magnesium ion and a monovalent cation, K+, NH4+, or Rb+, are required for catalytic activity. Polyphosphates and other common nucleotide triphosphates do not replace ATP as substrate. The stoichiometry of the catalytic reaction (Reaction 1) was established using 31P NMR, anaerobic molecular sieve chromatography, and radiochemical labeling procedures. ATP+selenide+H2O-->selenophosphate+Pi+AMP. In the absence of selenide, ATP is converted completely to AMP and orthophosphate upon prolonged incubation with elevated levels of enzyme. AMP is a competitive inhibitor of ATP, Ki = 170 microM, whereas selenophosphate and orthophosphate are weak inhibitors indicating a multistep reaction. Attempts to obtain direct evidence for a postulated enzyme-pyrophosphate intermediate using several experimental approaches are described. No exchange of [14C]AMP with ATP could be detected after the enzyme was freed of traces of contaminating adenylate kinase by chromatography on phenyl-Sepharose.

Biochemical analysis of Escherichia coli selenophosphate synthetase mutants. Lysine 20 is essential for catalytic activity and cysteine 17/19 for 8-azido-ATP derivatization.

A labile selenium donor compound, selenophosphate, is formed from selenide and ATP by selenophosphate synthetase. A cysteine residue (Cys-17) that is essential for catalytic activity of the enzyme (Kim, I.Y., Veres, Z., and Stadtman, T. C. (1992) J. Biol. Chem. 267, 19650-19654) is located in a glycine-rich segment near the N terminus of the protein. The possibility that this peptide sequence (HGAGCGCK) defines the ATP-binding site of the enzyme, as does a conserved ATP or GTP binding sequence (GXXXXGKS/T) found in several other proteins, was tested by site-specific mutagenesis. Thus His-13 and Gly-18 were changed to Asn and Val, respectively, and Lys-20 to Arg or Gln. Catalytic activity was markedly decreased by mutation of Lys-20 to Arg and abolished by mutation of Lys-20 to Gln. The mutation of Cys-19 and His-13 did not substantially alter the ATP Km and Vmax values, whereas the Gly-18 mutation resulted in a 4-fold increase in the ATP Km value compared with that of the wild type. ATP binding properties of the mutant enzymes were determined using Mn-[32P]ATP or Mn-[14C]ATP and gel filtration. Photoaffinity labeling of the proteins with [gamma-32P]8-azido-ATP showed that all mutant enzymes could be labeled with the ATP analog except those in which Cys-17 or Cys-19 were replaced with serine.

Monoselenophosphate: synthesis, characterization, and identity with the prokaryotic biological selenium donor, compound SePX.

A labile, selenium donor compound required for synthesis of selenium-dependent enzymes and seleno-tRNAs is formed from ATP and selenide by the SELD enzyme. This compound, tentatively identified as a selenophosphate [Veres, Z., Tsai, L., Scholz, T. D., Politino, M., Balaban, R. S., & Stadtman, T. C. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 2975-2979], is indistinguishable from chemically prepared monoselenophosphate by 31P NMR spectroscopy and ion pairing HPLC. Furthermore, addition of chemically prepared monoselenophosphate caused a dose-dependent decrease in the amount of 75Se incorporated into tRNAs from 75SePX generated in situ by SELD enzyme. A procedure is described for the chemical synthesis of monoselenophosphate in which the readily prepared (MeO)3PSe is converted in quantitative yield to (TMSO)3PSe followed by complete cleavage of the latter to monoselenophosphate in oxygen-free aqueous buffer. The chemical properties of chemically synthesized monoselenophosphate are described.

Escherichia coli mutant SELD enzymes. The cysteine 17 residue is essential for selenophosphate formation from ATP and selenide.

Synthesis of a labile selenium donor compound, selenophosphate, from selenide and ATP by the Escherichia coli SELD enzyme was reported previously from this laboratory. From the gene sequence, SELD is a 37-kDa protein that contains 7 cysteine residues, 2 of which are located at positions 17 and 19 in the sequence -Gly-Ala-Cys-Gly-Cys-Lys-Ile- (Leinfelder, W., Forchhammer, K., Veprek, B., Zehelein, E., and Böck, A. (1990) Proc. Natl. Acad. Sci. U.S.A. 73, 543-547). Inactivation of the enzyme by alkylation with iodoacetamide indicated that at least 1 cysteine residue in the protein is essential for enzyme activity. To test the possibility that the Cys17 and/or Cys19 residue might be essential, these were changed to serine residues by site-specific mutagenesis. The biological activities of the wild type and mutant proteins were studied using E. coli MB08 (selD-) transformed with plasmids containing the selD genes. The plasmid containing the Cys17-mutated gene failed to complement MB08, whereas the Cys19-mutated gene was indistinguishable from wild type. The mutant proteins, like the wild type enzyme, bound to an ATP-agarose matrix, showing that their affinities for ATP were unimpaired. Selenide-dependent formation of AMP from ATP was abolished by mutation of Cys17, but the Cys19 mutation had no effect on the ability of the enzyme to catalyze the reaction. These results indicate that Cys17 has an essential role in the catalytic process that leads to the formation of selenophosphate from ATP and selenide.

Synthesis of 5-methylaminomethyl-2-selenouridine in tRNAs: 31P NMR studies show the labile selenium donor synthesized by the selD gene product contains selenium bonded to phosphorus.

An enzyme preparation from Salmonella typhimurium catalyzes the conversion of 5-methylaminomethyl-2-thiouridine in tRNAs to 5-methylaminomethyl-2-selenouridine when supplemented with selenide and ATP. Similar preparations from a Salmonella mutant strain carrying a defective selD gene fail to catalyze this selenium substitution reaction. However, supplementation of the deficient enzyme preparation with the purified selD gene product (SELD protein) restored synthesis of seleno-tRNAs. In the absence of the complementary enzyme(s), the SELD protein catalyzes the synthesis of a labile selenium donor compound from selenide and ATP. 31P NMR studies show that among the products of this reaction are AMP and a compound containing selenium bonded to phosphorus. The reaction is completely dependent on the addition of both selenide and magnesium. The dependence of reaction velocity on ATP concentration shows sigmoidal kinetics, whereas dependence on selenide concentration obeys Michaelis-Menten kinetics indicating a Km value of 46 microM for selenide.

The effect of the 3'-OH group on the conformation and binding ability of anhydropyrimidine nucleosides to uridine phosphorylase.

2,2'-Anhydro-3'-deoxy-5-ethyluridine, a new pyrimidine nucleoside analog, has been examined in terms of its binding potency to uridine phosphorylase, and its conformation in solution (NMR) was studied. 2,2'-Anhydro-3'-deoxy-5-ethyluridine has a Ki value of 3.4 microM for uridine phosphorylase from rat intestinal mucosa. This value is approximately one order of magnitude lower than the Km for uridine (22 microM), the natural substrate. The presence of the 3'-OH group (in the ribo-configuration) on pyrimidine nucleoside analogs may not be considered a prerequisite for the binding to uridine phosphorylase; however, it enhances the binding in the case of flexible ligands cooperating in the process of conformation change toward a more favorable enzyme-ligand interaction. The presence of the 3'-OH group in pyrimidine nucleosides seems to be essential if the molecule is to become a substrate.

Metabolism of anti-herpes agent 5-(2-chloroethyl)-2'-deoxyuridine in mice and rats.

The enzymatic splitting and metabolic elimination of anti-viral agent 5-(2-chloroethyl)-2'-deoxyuridine [CEDU] have been studied. For elucidation of structures of metabolites, several different kinds of extraction, purification and spectroscopic methods were used (Extrelut LC, TLC, HPLC, MS, NMR, IR, UV and CD). For mass spectral analysis, various ionization techniques (EI, CI and FAB-MS) were performed as complementary methods. After oral administration of [14C]-CEDU to mice and rats, the parent compound, 5-(2chloroethyl) uracil [CEU] and hydroxylated CEU metabolites were isolated and identified from urine and faeces by the above mentioned methods. The CEDU showed rapid phosphorolysis in vitro with thymidine phosphorylase Km 41.0 +/- 5.0; and uridine phosphorylase Km 10.0 +/- 1.5. The cleavage of the N-glycosidic bond of the nucleoside analogue and a new metabolic pathway of CEDU [stereoselective oxidation of 5-(2-chloroethyl) uracil] was observed in both species.

cis-trans-isomerization of [E]-5-(2-bromovinyl)-2,2'-anhydrouridine in vivo in rats.

1. [E]-5-(2-bromovinyl)-2,2'-anhydrouridine [( E]BVANUR) has considerable antiviral activity against herpes simplex virus type 1 (HSV-1). 2. [E]BVANUR is not a substrate of pyrimidine nucleoside phosphorylases, but it is an inhibitor of uridine phosphorylase (Ki = 450 nM). 3. [E]BVANUR (trans-isomer, parent compound) undergoes isomerization to [Z]BVANUR (cis-isomer), the only metabolite in rat, which was identified by h.p.l.c., mass spectra and n.m.r. spectroscopy. 4. Absorption of the drug from the gastrointestinal tract after oral administration is minimal. Absorption of [E]BVANUR from the abdominal cavity after i.p. administration was slow.

Biosynthesis of selenium-modified tRNAs in Methanococcus vannielii.

Selenium-containing nucleosides are natural components of several tRNA species in Methanococcus vannielii. In the present study, the incorporation of selenium from 75SeO3(2-) into these macromolecules was investigated in sonic extracts of M. vannielii. Nucleoside analysis of the 75Se-labeled tRNAs from these in vitro reaction mixtures demonstrated that the selenium was present in 75Se-labeled nucleosides identical to the two naturally occurring 2-selenouridines produced in vivo. Incorporation of selenium into these nucleosides was ATP-dependent and was maximal after 20 min. Addition of O-acetylserine enhanced the activity 2- to 3-fold, implicating a role for selenocysteine in the reaction. Added L-selenocysteine could function as a selenium donor, but the D isomer and DL-selenomethionine were inactive. RPC-5 chromatography of bulk tRNA isolated from M. vannielii grown on 75SeO3(2-) separated five major species of seleno-tRNAs. The amino acid-accepting activity of these tRNAs was investigated.

In vitro incorporation of selenium into tRNAs of Salmonella typhimurium.

Broken-cell preparations of Salmonella typhimurium rapidly incorporated 75Se from 75SeO3(2-) into tRNA by an ATP-dependent process. Selenium incorporation in the presence of 50 microM 75SeO3(2-) (0.8-1 pmol per A260 unit) was enhanced by the selenocysteine precursor, O-acetyl-L-serine (to 3.7 pmol per A260 unit). This increase in incorporation was a function of O-acetyl-L-serine concentration. Neither O-acetyl-L-homoserine nor O-phospho-L-serine stimulated the incorporation of selenium into tRNA. The incorporation of 75Se from 75SeO3(2-) was decreased by adding L-selenocysteine but not by adding the D isomer. When homologous bulk tRNA was added to the broken-cell preparations, an increased rate of 75Se labeling was observed. The supernatant fraction of the broken-cell preparation contained all of the enzymes required for this process. Reversed-phase HPLC analysis of labeled bulk tRNA digested to nucleosides showed the presence of a labeled compound that coeluted with authentic 5-methylaminomethyl-2-selenouridine.

Differential effects of 2,2'-anhydro-5-ethyluridine, a uridine phosphorylase inhibitor, on the antitumor activity of 5-fluorouridine and 5-fluoro-2'-deoxyuridine.

2,2'-Anhydro-5-ethyluridine (ANEUR), a potent inhibitor of uridine phosphorylase, markedly potentiated the antitumor activity of fluorouridine (FUR) against murine mammary adenocarcinoma 755 in BDF1 mice and human colon adenocarcinoma LS174T in athymic-nude mice. Whereas ANEUR annihilated the antitumor activity of 5-fluoro-2'-deoxyuridine (FUdR) and 5'-deoxy-5-fluorouridine (DFUR) in the murine adenocarcinoma 755 system, it did not alter the antitumor activity of FUdR in the human adenocarcinoma LS174T system. In vitro, ANEUR proved inhibitory to the phosphorolytic cleavage of both FUR and FUdR by uridine phosphorylase, and this could explain why in vivo conversion of FUR and FUdR to 5-fluorouracil was suppressed. FUR can be held directly responsible for the antitumor effects observed in the murine adenocarcinoma 755 system, whereas in the activity against human adenocarcinoma LS174T may be mediated by both FUR and FUdR.

[The effect of 3,4-disuccinyldianhydrogalactitol, N-nitrosourea and their combination on DNA synthesis in normal and mouse P388 tumor cells]. / Vliianie 3,4-disuktsinildiangidrogalaktitola, N-nitrozomochevin i ikh kombinatsii na sintez DNK v normal'nykh i opukholevykh kletkakh myshei s leikozom P388.

The rates of incorporation of 2-14C-thymidine into DNA of leukemia P388, bone marrow, gastrointestinal mucosa and spleen cells at various time after administration of 3,4-disuccinyldianhydrogalactitol (DisuDAG), 1-methyl-1-nitrosourea (MNU), 1-(2-hydroxyethyl)-3-(2-chloroethyl)-3-nitrosourea (HECNU) and their combinations at different doses to mice with leukemia P388 (solid form) were studied. DisuDAG (80 mg/kg) induced the deep and the stable inhibition in DNA synthesis of leukemia P388, bone marrow and spleen cells. The combination of DisuDAG and HECNU at small doses induced the deep and the stable suppression of DNA synthesis in tumor cells, however DNA synthesis in normal dividing cells was shown to recover more rapidly than in leukemia P388 cells. Administration of the combination of DisuDAG with MNU to tumor-bearing mice induced more stable inhibition of DNA synthesis in tumor cells in comparison with MNU and DisuDAG. In vivo inhibition of DNA synthesis in leukemia P388 cells with DisuDAG and HECNU was not due to damage in pool of precursors (TCA soluble fraction).