Search PubMed⌕ Search

Biomedical subjects

G J Mulder

Publications and source records attributed to G J Mulder.

At least 73 records · Page 4Linked to original sources
PubMed1992

Fasting increases the susceptibility of rat hepatocytes to the cytotoxic effects of N-hydroxy-acetylaminofluorene. Effects on mitochondrial respiration and membrane potential.

Isolated rat hepatocytes were incubated with the carcinogen N-hydroxy-2-acetylaminofluorene (N-OH-AAF). Cells from fasted rats were much more susceptible to the cytotoxic effects of 1 mM N-OH-AAF than cells from fed rats: after approximately 90 min exposure the former were all dead but the latter still viable. Even after 240 min 25% of the "fed" cells were still viable. The loss of viability was preceded by a decrease in mitochondrial membrane potential (MMP) and inhibition of respiration; the mitochondrial respiration as measured in permeabilized cells appeared uncoupled. Addition of 15 mM fructose prevented cell death and the loss of MMP in cells both from fed and fasted rats to a large extent; however, uncoupling was not prevented. After incubation of hepatocytes from fasted rats with 1 mM [3H]N-OH-AAF for 120 min, 12 nmol [3H]N-OH-AAF became bound per mg cell protein. Addition of fructose decreased this to 7 nmol. In cells from fed animals 4 nmol [3H]N-OH-AAF became bound after 120 min, in this case fructose had no effect. Part of the protective effect of fructose might be explained by a decrease in intracellular ATP, which prevents the formation of reactive intermediates of N-OH-AAF resulting in a decrease of covalent binding, in addition, fructose protects via a yet to be determined mechanism.

Adenosine Triphosphate↗
PubMed1992

Involvement of intracellular Ca2+ and K+ in dissipation of the mitochondrial membrane potential and cell death induced by extracellular ATP in hepatocytes.

Isolated rat hepatocytes were incubated with extracellular ATP to induce a prolonged increase in intracellular Ca2+ ([Ca2+]i) and a loss of viability within 2 h. By using video-intensified fluorescence microscopy, the effects of exposure to extracellular ATP on [Ca2+]i, mitochondrial membrane potential (MMP) and cell viability were determined simultaneously in individual living hepatocytes. The increase in [Ca2+]i on exposure to ATP was followed by a decreasing MMP; there were big differences between individual cells. Complete loss of the MMP occurred before cell death was observed. Omission of K+ from the incubation medium decreased the cytotoxicity of ATP; under these conditions, intracellular K+ was decreased by more than 80%. Treatment with nigericin also depleted intracellular K+ and decreased ATP-induced toxicity. Protection against loss of viability by means of a decrease in intracellular [K+] was reflected by maintenance of the MMP. These observations suggest that ATP-induced cell death may be caused by a mechanism that has been described for isolated mitochondria: after an increase in Ca2+ levels, a K+ influx into mitochondria is induced, which finally disrupts the MMP and leads to cell death.

Adenosine Triphosphate↗
PubMed1992

Stereoselective conjugation of 2-bromocarboxylic acids and their urea derivatives by rat liver glutathione transferase 12-12 and some other isoforms.

Glutathione (GSH) conjugation of the separate enantiomers of five 2-bromocarboxylic acids and some of their urea derivatives by rat liver GSH transferases (GSTs) was studied. The liver cytosolic fraction conjugated all compounds, except for (R)-2-bromoisovaleric acid, with a variable degree of stereoselectivity. A GST pool, prepared by S-hexyl-GSH affinity chromatography, conjugated the urea derivatives at a somewhat higher rate but had very little activity towards the carboxylic acids, indicating that much activity towards the latter substrates was due to transferases not bound by the affinity column. Therefore, the activity was studied of some pure GSTs that are bound only slightly by the affinity column towards the separate enantiomers of 2-bromovaleric acid (BV), its urea derivative and 2-bromo-3-phenylpropionic acid (BPP). No activity was detected with transferases 5-5 and 8-8. Transferase 1-1 was active towards all compounds with high activity towards the urea derivatives. Transferase 12-12 showed high, stereospecific activity towards the R enantiomers of BV, its urea derivative and BPP.

Animals↗
PubMed1992

Selective and potent inhibition of different hepatic UDP-glucuronosyltransferase activities by omega,omega,omega-triphenylalcohols and UDP derivatives.

A homologous series of omega,omega,omega-triphenylalcohols and corresponding omega,omega,omega-triphenylalkyl-UDP derivatives was synthesized and tested as inhibitors of UDP-glucuronosyltransferase (UGT) activity in rat liver microsomes, with 1-naphthol, testosterone and bilirubin as substrates. Introduction of the UDP moiety in the triphenylalcohols increased their inhibition potency markedly toward the isoforms which glucuronidate 1-naphthol and testosterone, but strongly decreased that toward bilirubin. The inhibiting potency of the UDP-derivatives increased as a function of the length of the hydrocarbon chain. The best inhibitor 7,7,7-triphenylheptyl-UDP showed an I50 of 30 and 10 microM for 1-naphthol and testosterone glucuronidation, respectively; even a 1 mM concentration of the compound had little, if any, effect on bilirubin glucuronidation. The inhibition by 7,7,7-triphenylheptyl-UDP was mixed-type toward 1-naphthol, and non competitive toward testosterone (apparent K(i) 30 microM and 1.7 microM, respectively); on the other hand, the inhibition was competitive toward the common substrate UDP-glucuronic acid (apparent K(i) 1.9-1.2 microM). In addition, 7,7,7-triphenylheptyl-UDP (0.25-0.50 mM) almost inhibited glucuronidation of 1-naphthol and testosterone catalyzed by the recombinant rat liver UGT-2B1 and human liver UGT-1A1, whose cDNA has been expressed in V79 cells. In conclusion, the data indicate that 7,7,7-triphenyheptyl-UDP interacted competitively with the UDP binding site of UGT. The results also indicate that it is possible to design transition state analogue inhibitors with specificity for different UGT forms.

Alcohols↗
PubMed1992

Relationship between glutathione content in liver and glutathione conjugation rate in the rat in vivo. Effect of buthionine sulphoximine pretreatment on conjugation of the two 2-bromoisovalerylurea enantiomers during intravenous infusion.

The relationship between hepatic glutathione content and hepatic glutathione conjugation rate in the rat in vivo was investigated. As substrate for glutathione conjugation, racemic (R,S)-2-bromoisovalerylurea (BIU) was used which gives rise to the biliary excretion of two diastereoisomeric glutathione conjugates and the urinary excretion of two diastereoisomeric mercapturates. The excretion rate of the glutathione conjugate in bile reflects hepatic conjugation exclusively. An intravenous infusion of BIU was given and the excretion rates of the metabolites in bile and urine were determined. The glutathione concentration in the liver was followed by taking biopsies every hour. Glutathione was depleted by the infused substrate; in rats that were pretreated with the inhibitor of glutathione biosynthesis, buthionine sulphoximine (BSO), the depletion of the glutathione content was more rapid. The rate of excretion of the glutathione conjugate in bile was plotted against hepatic glutathione content. These results indicate that the 'organ Km' for glutathione in the liver is approximately 0.5 mumol/g of liver, so that the hepatic glutathione conjugation rate is decreased only at severe glutathione depletion.

Acetylcysteine↗
PubMed1992

Immobilization of solubilized UDP-glucuronosyltransferase from rat liver microsomes to Sepharose 4B.

A method for the covalent binding of rat liver UDP-glucuronosyltransferase to a cyanogen bromide-activated agarose matrix is described. The rat liver microsomal fraction was solubilized with 8 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS); 90% of the microsomal protein was solubilized. Some 50-60% of this protein became bound covalently to the activated agarose matrix. The immobilized UPD-glucuronosyltransferase remained completely active for 50 days when stored at 4 degrees in a 20% (v/v) glycerol buffer (pH 7.4). The immobilized enzyme has a temperature optimum around 37 degrees, and a broad pH optimum (pH 5-7.4). The enzyme displayed linear kinetics over a period of 1 hr; it conjugates a large variety of substrates.

Animals↗
PubMed1992

Stereospecific glutathione conjugation of (R)- and (S)-2-bromoisovalerylurea in freshly isolated rat kidney proximal tubular cells.

The glutathione conjugation of 2-bromoisovalerylurea (BIU) was studied in isolated proximal tubular kidney cells of the rat. Racemic (R,S)-BIU was incubated with the cell suspension, and the incubation medium was analysed for the diastereomeric glutathione (GSH) conjugates, cysteine conjugates and mercapturates that can be formed from (R)- and (S)-BIU. Only the mercapturate formed from (R)-BIU was found, as well as its cysteine precursor. No GSH conjugates were detected. These results indicate that these cells conjugate only the (R)-BIU enantiomer, and that the GSH conjugate is immediately further metabolized to its cysteine conjugate and mercapturate.

Acetylcysteine↗
PubMed1992

Enantioselective determination of R- and S-(alpha-bromoisovaleryl)urea in plasma using high-performance liquid chromatography after solid-phase extraction.

A stereoselective method has been developed for the determination of R- and S-(alpha-bromoisovaleryl)urea in plasma and saliva after oral administration. The chiral separation was carried out on Chiralcel OJ or OD columns with hexane--2-propanol as the mobile phase. The poor detection properties of the analyte required the development of an effective sample pretreatment procedure to enable ultraviolet detection at 210 nm. Solid-phase extraction using hydrophobic Amberlite XAD-2 in combination with washing steps at alkaline and acidic pH completely removed interfering components of the biological matrix and allowed the detection of the optical isomers at concentrations down to 10 ng/ml (0.05 microM). The method was validated by determining the recovery, linearity, accuracy and within-day and between-day repeatability at 50, 200 and 2000 ng/ml. Application to the analysis of plasma and saliva samples is demonstrated.

Bromisovalum↗
PubMed1992

Glutathione conjugation and pharmacokinetics of 2-bromo-3-phenylpropionic acid in vitro and in the rat in vivo.

Glutathione (GSH) conjugation of the chiral compound 2-bromo-3-phenylpropionic acid (BPP) was studied in vitro and in the rat in vivo. GSH conjugation of BPP, catalyzed by a mixture of glutathione-S-transferases (GST's) from rat liver cytosol in vitro, was stereoselective: at a substrate concentration of 250 microM, (R)-BPP was more rapidly conjugated than (S)-BPP (R/S-ratio = 2.6). The blood elimination kinetics of the separate BPP enantiomers and the biliary excretion kinetics of the corresponding GSH conjugates were studied in the rat in vivo after administration of (R)- or (S)-BPP at a dose level of 50 mumol/kg. Elimination of (R)-BPP from blood was faster than that of (S)-BPP: half lives were 9 +/- 2 min for (R)-BPP and 13 +/- 1 min for (S)-BPP. The biliary excretion rate of the GSH conjugate of (R)-BPP declined monoexponentially, while that of the GSH conjugate of (S)-BPP displayed a biphasic profile. Half lives of excretion were 13 +/- 1 for the GSH conjugate of (R)-BPP, and 11 +/- 2 for the GSH conjugate of (S)-BPP (second phase). The first phase in the biliary excretion of the GSH conjugate of (S)-BPP could not be attributed to capacity limitation of biliary transport carriers as higher excretion rates were attained upon administration of higher doses (100 and 200 mumol/kg) of (S)-BPP). The blood elimination profiles of (R)- and (S)-BPP differed greatly from the biliary excretion profiles of the corresponding GSH conjugates. This suggests that the kinetics of BPP conjugate excretion are determined by other processes than hepatic GSH conjugation.

Animals↗
PubMed1992

Noninvasive in vivo 13C-NMR spectroscopy of a 13C-labeled xenobiotic in the rat.

This study demonstrates that the xenobiotic product, 1-(o-chlorophenyl)-1-(p-chlorophenyl)-2,2-dichloro-3-13C-propane can be monitored in the liver of an intact animal by in vivo 13C surface coil NMR spectroscopy after intraperitoneal administration. The carbon-13 label could be detected after a single dose of only 200 mg/kg of the product. The intrahepatic changes of the signal intensity of the labeled product were monitored as a function of time. No signals corresponding to metabolites could be detected.

Animals↗
PubMed1992

Sulfation of hydroxylamines and hydroxamic acids in liver cytosol from male and female rats and purified aryl sulfotransferase IV.

Sulfation activity towards hydroxamic acids and hydroxylamines was determined in liver cytosols for juvenile and adult males and female rats, as well as in purified rat liver aryl sulfotransferase IV preparations. Sulfation activity towards the hydroxamic acids N-hydroxy-2-acetylaminofluorene, N-hydroxy-2-acetylaminophenanthrene, N-hydroxy-4-acetylaminobiphenyl, N-hydroxy-4'-fluoro-4-acetylaminobiphenyl, N-hydroxy-2-acetylamino-5-phenylpyridine, was higher in cytosols derived from adult males (two or three times) than in those from adult females and juveniles (both sexes). N-Hydroxy-2-acetylamino-3-methyl-5-phenylpyridine (N-OH-2AAMPP), however, was poorly sulfated by any of the cytosols. Sulfation activity towards the hydroxylamines N-hydroxy-2-aminofluorene, N-hydroxy-2-aminophenanthrene, N-hydroxy-4-aminobiphenyl, N-hydroxy-4'-fluoro-4-aminobiphenyl was much lower. N-Hydroxy-2-amino-5-phenylpyridine (N-OH-2APP), however, was sulfated much better than the other hydroxylamines. No higher sulfation activity in adult male cytosols for hydroxylamines was found, except for N-OH-2APP and N-hydroxy-2-amino-3-methyl-5-phenylpyridine (N-OH-2AMPP). Purified aryl sulfotransferase IV (AST IV) converted all hydroxamic acids; N-OH-2AAMPP was a poor substrate. Of the hydroxylamines only N-OH-2APP and N-OH-2AMPP were conjugated. These results suggest that hydroxylamines and hydroxamic acids are converted by different sulfotransferases in the rat in vivo. They also indicate that AST IV may be the major enzyme responsible for sulfation of a variety of aromatic hydroxamic acids in the male rat liver. The results presented here are discussed in relation to the carcinogenic effects of some of these compounds.

Aging↗
PubMed1992

Photoactivation of 2-nitrofluorene in vitro and in the rat in vivo. UVA-induced formation of reactive intermediates that bind covalently to RNA and protein.

2-Nitrofluorene (2-NF), an environmental pollutant, can be activated by UV light to reactive intermediates that bind covalently to RNA and protein in vitro: high levels of covalent binding were obtained. This covalent binding was not dependent on the presence of oxygen in the solution and could be decreased by glutathione. Hydrolysis of the in vitro modified RNA and subsequent analysis of the liberated bases by HPLC revealed that approximately 15% of the covalent binding of 2-NF to RNA could be attributed to the formation of a guanosine adduct of nitroreduced 2-NF, N-(deoxyguanosin-8-yl)-2-aminofluorene. Many other adducts are formed of which the structure and mechanism of formation are as yet unknown. The possibility of photoactivation of 2-NF in the rat in vivo was also investigated. Photoactivation increased covalent binding of 2-NF in the skin but not in other organs. The mechanism of the photoactivation of 2-NF is discussed.

Animals↗
PubMed1992

Chiral inversion and stereoselective glutathione conjugation of the four 2-bromo-3-methylvaleric acid stereoisomers in the rat in vivo and in vitro.

Glutathione conjugation of the four 2-bromo-3-methylvaleric acid (BMV) stereoisomers was studied in vitro (rat liver cytosol) and in the rat in vivo (by monitoring biliary excretion of the glutathione conjugates). Rat liver cytosol catalyzed the formation of the corresponding glutathione conjugates in a ratio of 28:7:1:0 for the isomers 2S,3S-, 2S,3R-, 2R,3R- and 2R,3S-BMV, respectively. In the rat in vivo, a similar rank order was found: no conjugation of the 2R,3S isomer, whereas the biliary excretion half-lives of the GSH conjugates of the 2S,3S-, 2S,3R- and 2R,3R-isomers were 11, 36 and 70 min, respectively. These results show that isomers with the C2 carbon in the S configuration are more rapidly conjugated than those with the R configuration, and that the chiral center at the C3 carbon atom affects the conjugation rate at the C2 carbon. In addition to the SN2-type glutathione conjugates, from three substrates the glutathione conjugate of the corresponding diastereomer was formed, indicating bidirectional chiral inversion at the C2 carbon atom of the isomer. For instance, 2S,3R-BMV yielded both 2R,3R-MV-G and 2S,3R-MV-G. The biliary excretion half-lives of the "inverted" conjugates formed from the 2R,3S-, 2R,3R- and 2S,3R-isomer were 54 +/- 3, 75 +/- 3 and 38 +/- 3 min, respectively.

Animals↗
PubMed1991

Inhibition of glutathione S-transferase 3-3 by glutathione derivatives that bind covalently to the active site.

In all, 13 GSH derivatives have been synthesized and tested for their potency to inhibit glutathione S-transferase (GST) 3-3. All of these derivatives contained a reactive group that could potentially react with the enzyme active site. Best results were obtained with the phenylthiosulphonate derivative of GSH, GSSO2Ph. Preincubation of GST 3-3 with a 100 microM concentration of this inhibitor resulted in a time-dependent loss of activity: after 30 min at pH 6.5 and 25 degrees C, 51% of the activity was lost. At more alkaline pH, the activity is more rapidly inhibited: at pH 8.0 the 90%-inhibition level is already reached after 10 min preincubation. Separation of enzyme and excess unbound GSSO2Ph after preincubation by gel-filtration chromatography did not result in a reappearance of enzyme activity. If 100 microM-GSH was added to the preincubation mixture at pH 7.4, inhibition was almost completely prevented. Addition of S-(hexyl)glutathione (20 microM) could delay the inhibition but, ultimately, not prevent it. The inhibited enzyme could be re-activated by addition of 10 mM-2-mercaptoethanol: 60 min after this thiol was added, the inhibited GST-3- activity was bacxk to the control level. GSH at the same concentration could not re-activate the enzyme. On the basis of these results, on the known reactivity of thiosulphonate compounds, and on current knowledge about the amino acid residues involved in GST catalysis, a covalent modification of an active-site cysteine residue by mixed-disulphide formation between enzyme and the cosubstrate GSH is postulated. Information on the synthesis and characterization of the GSH derivatives is given in Supplementary Publication SUP 50166 (5 pages) which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1991) 273, 5.

Amino Acid Sequence↗
PubMed1991

Studies on the activity and activation of rat liver microsomal glutathione transferase with a series of glutathione analogues.

The substrate specificity of rat liver microsomal glutathione transferase toward glutathione has been examined in a systematic manner. Out of a glycyl-modified and eight gamma-glutamyl-modified glutathione analogues, it was found that four (glutaryl-L-Cys-Gly, alpha-L-Glu-L-Cys-Gly, alpha-D-Glu-L-Cys-Gly, and gamma-L-Glu-L-Cys-beta-Ala) function as substrates. The kinetic parameters for three of these substrates (the alpha-D-Glu-L-Cys-Gly analogue gave very low activity) were compared with those of GSH with both unactivated and the N-ethylmaleimide-activated microsomal glutathione transferase. The alpha-L-Glu-L-Cys-Gly analogue is similar to GSH in that it has a higher kcat (6.9 versus 0.6 s-1) value with the activated enzyme compared with the unactivated enzyme but displays a high Km (6 versus 11 mM) with both forms. Glutaryl-L-Cys-Gly, in contrast, exhibited a similar kcat (8.9 versus 6.7 s-1) with the N-ethylmaleimide-treated enzyme but retains a higher Km value (50 versus 15 mM). Thus, the alpha-amino group of the glutamyl residue in GSH is important for the activity of the activated microsomal glutathione transferase. These observations were quantitated by analyzing the changes in the Gibbs free energy of binding calculated from the changes in kcat/Km values, comparing the analogues to GSH and each other. It is estimated that the binding energy of the alpha-amino group of the glutamyl residue in GSH contributes 9.7 kJ/mol to catalysis by the activated enzyme, whereas the corresponding value for the unactivated enzyme is 3.2 kJ/mol. The importance of the acidic functions in glutathione is also evident as shown by the lack of activity with 4-aminobutyric acid-L-Cys-Gly and the low kcat/Km values with gamma-L-Glu-L-Cys-beta-Ala (0.03 and 0.01 mM-1s-1 for unactivated and activated enzyme, respectively). Utilization of binding energy from a correctly positioned carboxyl group in the glycine residue (10 and 17 kJ/mol for unactivated and activated enzyme, respectively) therefore also appears to be required for optimal activity and activation. A conformational change in the microsomal glutathione transferase upon treatment with N-ethylmaleimide or trypsin, which allows utilization of binding energy from the alpha-amino group of GSH as well as the glycine carboxyl in catalysis, is suggested to account for at least part of the activation of the enzyme.

Amino Acid Sequence↗