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G J Mulder

Publications and source records attributed to G J Mulder.

At least 145 records · Page 8Linked to original sources
PubMed1984

Inhibition of glutathione efflux in the recirculating rat liver perfusion by cysteine but not by oxothiazolidine carboxylate, an intracellular cysteine precursor.

In the recirculating rat liver perfusion a continuous release of glutathione into the perfusion medium is observed. Addition of L-cysteine to the perfusion medium immediately arrested this glutathione efflux. The cysteine precursor oxothiazolidine carboxylate did not block the glutathione efflux in spite of the fact that it generated more L-cysteine inside the liver cells than L-cysteine itself; L-cysteine is rapidly oxidized to cystine, that is no longer taken up by the liver. The results suggest that the inhibition of glutathione efflux results from the presence of cystine in the perfusion medium.

Animals↗
PubMed1984

Extrahepatic sulfation and glucuronidation in the rat in vivo. Determination of the hepatic extraction ratio of harmol and the extrahepatic contribution to harmol conjugation.

The phenolic compound, harmol, is metabolized by sulfation and glucuronidation in the rat in vivo. In the present study, various harmol infusion rates into the jugular vein were used to delineate first-order conditions whereby total body clearance was maximal and constant; at low infusion rates the steady state harmol concentration in blood varied proportionally with the infusion rate. At infusion rates of 167 nmole/min and below, the steady state clearance of harmol was 60 ml/min or 200 ml/min/kg. Because this value for total body clearance greatly exceeded the value for hepatic blood flow rate (20 ml/min for a 300 g rat), considerable extrahepatic conjugation of harmol was suggested. At higher harmol infusion rates the total clearance decreased. Since an intraportal infusion of 167 nmole/min to the rat yielded, during steady state, the same arterial harmol blood concentration as a 52 nmole/min jugular infusion, the hepatic extraction ratio of harmol in vivo was 0.7. Extrahepatic clearance, therefore, constituted about 77% of total body clearance (after taking the difference between total body clearance and hepatic clearance). Total sulfation clearance was 52 ml/min, and greatly exceed the value for hepatic clearance (14 ml/min). Extrahepatic clearance for sulfation (at least 38 ml/min) therefore accounted for a major proportion of the sulfation activity. Blood platelets did not seem to contribute to sulfation or glucuronidation in vivo.

Alkaloids↗
PubMed1984

D-cysteine as a selective precursor for inorganic sulfate in the rat in vivo. Effect of D-cysteine on the sulfation of harmol.

D-Cysteine, the unphysiological isomer of the sulfur containing amino acid L-cysteine, is not utilized for protein synthesis, glutathione synthesis or taurine production; it was tested as a selective precursor for inorganic sulfate, required for sulfation of xenobiotics. Both cysteine isomers were injected intravenously in the rat, in order to investigate their sulfoxidation to inorganic sulfate. The rates of sulfoxidation were very similar, so that stereospecificity for the amino acid seemed not to play a role. When the rats were fed a low-protein diet (LP-diet; containing only 8% casein as source of amino acids) the serum sulfate concentration decreased to about 20% of control. Under these circumstances the rate of sulfoxidation of both isomers was decreased to the same extent. In order to confirm that both cysteine isomers were equally efficient in providing inorganic sulfate for sulfation of xenobiotics, a constant infusion of harmol (a substrate for sulfation) was given to rats fed the LP-diet. Administration of L- or D-cysteine yielded similar increases in sulfation of harmol under these conditions. These results show that D-cysteine can be used to selectively enhance sulfate availability.

Alkaloids↗
PubMed1984

Reaction of mutagenic phenacetin metabolites with glutathione and DNA. Possible implications for toxicity.

The direct-acting mutagens, N-hydroxy-p-phenetidine and p-nitrosophenetole, are known to be metabolites of the analgesic phenacetin and may be responsible for its carcinogenic activity. In this study, the potential detoxification of these metabolites by glutathione was examined. Glutathione reacted rapidly with p-nitrosophenetole, which was quantitatively converted to a single product as determined by high-pressure liquid chromatography. The analysis of the product by fast atom bombardment mass spectrometry and 500-MHz 1H-NMR spectroscopy established its structure as N-(glutathion-S-yl)-p-phenetidine. The same glutathione conjugate was also formed when N-hydroxy-p-phenetidine was incubated with glutathione. However, since conjugate formation from N-hydroxy-p-phenetidine occurred slowly and was decreased in the presence of an argon atmosphere as well as by higher levels of glutathione, it was concluded that the conjugate resulted from oxidation of the N-hydroxy arylamine to the nitrosoarene, which subsequently reacted with glutathione. N-(Glutathion-S-yl)-p-phenetidine was semistable in water (half-life, 6-7 hr) and very unstable in the presence of nucleophiles such as 10 mM glutathione (half-life, 7 min), quantitatively decomposing to p-phenetidine. The conjugate was also very unstable in acidic buffers (half-life, 17 min, pH 5). Radiolabeled N-hydroxy-p-phenetidine, but not p-nitrosophenetole, was shown to bind covalently to calf thymus DNA in vitro, and 4 times more binding was detected at pH 5 than at pH 7. Glutathione did not significantly decrease binding of the N-hydroxy derivative at either pH, nor did purified ring-radiolabeled N-(glutathion-S-yl)-p-phenetidine significantly bind to DNA at either pH. Thus, we hypothesize that an important detoxification pathway for phenacetin in vivo could involve the facile oxidation of N-hydroxy-p-phenetidine to p-nitrosophenetole, which then reacts rapidly with glutathione to form an excretable conjugate.

Aminophenols↗
PubMed1983

A simple method of obtaining multiple blood samples from the portal vein and the hepatic vein in the rat in vivo.

A very simple and rapid technique for inserting a catheter in the portal vein and the hepatic vein in the anesthesized rat in vivo is described. The pointed, saline-containing PE tubing is frozen in liquid nitrogen, whereupon it is used as a 'needle' to insert the catheter into the blood vessel. Multiple blood samples can be obtained from the portal and the hepatic vein at the same time, so that in situ extraction of drugs by the liver can be measured in vivo, since hepatic blood flow is uninterrupted.

Animals↗
PubMed1983

Use of pentachlorophenol as long-term inhibitor of sulfation of phenols and hydroxamic acids in the rat in vivo.

Inhibition of sulfation of the phenolic compound harmol (7-hydroxy-1-methyl-9H-pyrido[3,4-b]indole) by pentachlorophenol (PCP) was studied in the Wistar rat: PCP was administered in various ways to find a convenient method for long-term inhibition of sulfation. High doses of PCP or sodium pentachlorophenolate (NaPCP) in the diet (350 ppm) or NaPCP in the drinking water (1.4 mM) of Wistar rats for one week inhibited the sulfation of harmol by 30-45%. The plasma concentration of PCP in rats with NaPCP (1.4 mM) in their drinking water was highest (270 microM) in the period that the animals were kept in the dark and consumed food and water. This is explained by a rapid elimination: the elimination of PCP from plasma, after intravenous administration, showed a biphasic disappearance curve with half-lives of 2.17 and 7.24 hrs, respectively. This is much faster than in Sprague-Dawley rats. A log-linear correlation was found between the plasma concentration of pentachlorophenol and the inhibition of harmol sulfation. Although administration of NaPCP to rats in their drinking water inhibited the sulfation of harmol only by 45%, it inhibited the sulfation of the carcinogenic arylhydroxamic acid N-hydroxy-2-acetylaminofluorene by 70-75%.

Animals↗
PubMed1983

Effects of variation in the dietary supply of cysteine and methionine on liver concentration of glutathione and "active sulfate" (PAPS) and serum levels of sulfate, cystine, methionine and taurine: relation to the metabolism of acetaminophen.

Amounts of the sulfur-containing amino acids methionine and cysteine in synthetic diets were decreased from 22.8 mmol methionine (= 100 Met) and 26.4 mmol cysteine (= 100 Cys) per kilogram diet in the control group to (Met/Cys) 100:0, 75:0, 50:0, 25:0 and 25:25, respectively, in experimental diet groups, in order to evaluate effects of limiting sulfur supply on the availability of cosubstrates for conjugation. Below a Met level of 22.8 mmol/kg growth rates decreased. Urinary excretion of inorganic sulfate decreased to 10-20% of control values in all groups. Feeding diets 100:0 and 75:0 for 14 days resulted in a decrease of the concentration of inorganic sulfate in serum; on diets 25:25 and 25:0 an increase was found. A decreased content of methionine/cysteine resulted in an increase in cystine, a decrease in taurine and no change in methionine concentration in serum. In all experimental groups the glutathione concentration in the liver diminished to about 20% of control values, and the hepatic concentration of "active sulfate" (PAPS: adenosine 3'-phosphate 5'-phosphosulfate) decreased in most rats. At the lowest methionine supply, sulfate conjugation of acetaminophen decreased to 50% of control. The formation of the acetaminophen glutathione conjugate remained unaffected, in spite of a decreased hepatic glutathione availability.

Acetaminophen↗
PubMed1983

Sulfation and glucuronidation as competing pathways in the metabolism of hydroxamic acids: the role of N,O-sulfonation in chemical carcinogenesis of aromatic amines.

Aromatic amines can be metabolized by N-acetylation and N-hydroxylation to hydroxamic acids; these subsequently are conjugated to form the N,O-sulfonate and N,O-glucuronide conjugates. The N,O-sulfonates are highly labile metabolites that generate reactive intermediates involved in the covalent binding of the parent compound to protein, RNA and DNA, as well as to low molecular compounds like glutathione. This paper discusses methods used to decrease sulfation in vivo, and thereby to enhance the formation of the more stable N,O-glucuronides from N-hydroxy-2-acetylaminofluorene and N-hydroxy-4-acetylamino-4'-fluorobiphenyl. Acetaminophen pretreatment decreases the sulfate availability, but results in many side effects that complicate the analysis of the results. An 8% casein diet reduces the sulfate availability in the rat to approximately 20% of control and thus offers an effective approach to decrease sulfation. The most effective selective inhibition of sulfation is by pentachlorophenol, which very strongly reduces N,O-sulfonation of both hydroxamic acids, and selectively inhibits the formation of DNA adducts that have retained the N-acetyl group. This inhibitor and the related 2,6-dichloro-4-nitrophenol can be employed to study the role of sulfation of hydroxamic acids in initiation and promotion of tumor formation by aromatic amines.

Acetaminophen↗
PubMed1983

Normal and retrograde perfusion to probe the zonal distribution of sulfation and glucuronidation activities of harmol in the perfused rat liver preparation.

The role of zonal distributions of metabolic activities (sulfation and glucuronidation) in the liver on the kinetics of harmol conjugation was investigated. A computer simulation approach was adopted to better understand the effects of distributions of these conjugation activities in competition for a common drug substrate. Several distributions of the sulfation and glucuronidation systems were defined with respect to the flow path of blood; the conjugation activities along the flow path were in turn translated as time elapsed after entry of the substrate via blood into the organ. Realistic values of Km and Vmax for the sulfation and glucuronidation systems were assigned in the simulations. Directional flow, namely, normal vs. retrograde delivery of substrate was used as an additional variable. When the "center" of distribution of the sulfation system was anterior to that for glucuronidation, the steady-state hepatic extraction ratio of harmol (E) would increase, whereas the ratio of the steady-state rates of formation of harmol sulfate to harmol glucuronide (S/G ratio) would decrease when harmol was presented in a reversed direction (via retrograde perfusion), as compared with normal directional flow to the liver. The converse was true for an anterior distribution of the glucuronidation system; E would decrease, whereas S/G would increase with retrograde perfusion. To evaluate such zonal differences experimentally, perfusion studies were conducted in livers of male Wistar rats. A constant concentration of harmol (50 microM) was delivered under constant hepatic flow rate (10 ml/min/liver) by normal and retrograde perfusions to the same rat liver preparation. The steady-state hepatic E was higher (P less than .005) during retrograde perfusion than during normal perfusion, whereas the S/G ratio was significantly decreased (P less than .0005). The observations suggest an intercellular difference in the distribution of the two conjugating systems and are consistent with the view of the center of distribution for the sulfation system being anterior to the center of distribution for the glucuronidation system along the normal flow path of blood in the liver.

Alkaloids↗
PubMed1982

The dependence of the rate of sulphate conjugation on the plasma concentration of inorganic sulphate in the rat in vivo.

The dependence of the rate of sulphation of harmol [7-hydroxy-1-methyl-9H-pyrido(3,4-b)indole] on the plasma concn of inorganic sulphate was estimated in the rat in vivo. This was done in rats that were fed a low protein diet for 4 days to deplete the pool of inorganic sulphate, which decreased the plasma concn of sulphate from 0.9 mM normally to about 0.15 mM. Infusion of sodium sulphate at a rate that was stepwise increased, yielded a range of constant plasma sulphate levels below and above the physiological concn in plasma. By a constant infusion of harmol (6 mumoles/min/kg body wt), a steady state in the excretion of harmol sulphate could be reached at various plasma concns of sulphate. The apparent Km for inorganic sulphate was approx 0.3 mM. The relevance of these data in the evaluation of the effects of changes in the availability of inorganic sulphate on sulphate conjugation of xenobiotics is discussed.

Animals↗
PubMed1982

Kinetics of sulfation and glucuronidation of harmol in the perfused rat liver preparation. Disappearance of aberrances in glucuronidation kinetics by inhibition of sulfation.

Harmol is conjugated by glucuronidation and sulfation when it is given to the rat in vivo. In the once-through perfused rat liver preparation glucuronidation of harmol shows kinetic aberrances [Pang et al., J. Pharmac. exp. Ther. 219, 134 (1981)]. In order to further delineate the mechanism behind this, sulfation was inhibited to about 10% of control by 2,6-dichloro-4-nitrophenol. The loss of sulfation was compensated by an increase in the rate of glucuronidation, keeping the total clearance by the liver virtually constant in spite of the loss of sulfation. The inhibition of sulfation eliminated the previously observed lag-phase in the kinetics of glucuronidation; the rate of glucuronidation was now almost linear with the input concentration of the substrate harmol. The constant clearance of harmol in spite of inhibition of sulfation, the occurrence of the lag-phase in glucuronidation in the presence of sulfation, and the disappearance of this lag-phase in the absence of sulfation can be explained by either diffusion-limited metabolism of harmol or a heterogeneous sub-lobular distribution of the sulfating and glucuronidating systems. Activation of glucuronidation by harmol at high concentration can be excluded.

Alkaloids↗
PubMed1982

Selective inhibition of sulfate conjugation in the rat: pharmacokinetics and characterization of the inhibitory effect of 2,6-dichloro-4-nitrophenol.

The pharmacokinetics of 2,6-dichloro-4-nitrophenol (DCNP) have been studied in the rat. Upon i.v. injection the plasma decay curve of DCNP showed a rapid distribution phase. After 30 min the plasma concentration reached a value that was constant for at least 90 min, indicating very slow elimination of DCNP. The volume of distribution was 88 ml/kg and a high degree of binding (over, 99%) of DCNP in vitro to bovine serum albumin was found. The concentration of DCNP in the liver was between 30 and 50% of the plasma values. While in vivo the effect of DCNP persisted for a long time, its action was readily reversible in the single-pass perfused rat liver. In vivo, the effect of the dose of DCNP on the inhibition of sulfation of the phenolic compound harmol was investigated. Upon the i.v. injection of 26 mumole DCNP/kg an instantaneous and complete inhibition of sulfation of harmol was found. Using this property of DCNP, the rate of sulfation of harmol in vivo was evaluated in relation to the dose and the time after injection of the substrate. Saturation of sulfation apparently occurred because the consumption of inorganic sulfate was extremely small.

Animals↗
PubMed1982

Identification of glutathione conjugates formed from N-hydroxy-2-acetylaminofluorene in the rat.

Rats which had received N-hydroxy-2-acetylaminofluorene (N-OH-AAF) excreted two glutathione conjugates in the bile. Pretreatment with the sulphotransferase inhibitor pentachlorophenol reduced the amounts excreted by 50% suggesting that these conjugates were formed in vivo from the highly reactive N-O-sulphate ester of N-OH-AAF (AAF-N-sulphate). In order to identify these biliary conjugates synthetic GSH conjugates were prepared by the reaction of N-acetoxy-AAF, (a model compound for AAF-N-sulphate), with GSH. Four conjugates were isolated and characterized by ultraviolet, proton magnetic resonance, mass spectroscopy and by the determination of their amino acid composition. These conjugates were identified as 1-, 3-, 4- and 7-(glutathion-S-yl)-N-acetyl-2-aminofluorene, respectively. The two biliary conjugates were identified as 1- and 3-(glutathion-S-yl)-N-acetyl-2-aminofluorene.

2-Acetylaminofluorene↗
PubMed1982

Formation and identification of glutathione conjugates from 2-nitrosofluorene and N-hydroxy-2-aminofluorene.

2-Nitrosofluorene (NOF) and N-hydroxy-2-aminofluorene (N-HO-AF) are potent direct-acting mutagens, derived from metabolic activation of the carcinogen, N-acetyl-2-aminofluorene (AAF). To assess the ability of cellular glutathione (GSH) to detoxify these electrophilic derivatives, we examined the reaction of NOF and N-HO-AF with GSH in vitro. Two reaction products were isolated and identified as glutathionyl derivatives of 2-aminofluorene (AF) containing an N-S linkage. Amino acid analysis, infrared and NMR (500 MHz) spectroscopy, fast atom bombardment mass spectrometry and analysis of reaction characteristics and hydrolysis products established their structures as N-(glutathion-S-yl)-2-aminofluorene S-oxide (GS-AFI) and N-(glutathion-S-yl)-2-aminofluorene (GS-AFII). Ascorbic acid, which reduces NOF to N-HO-AF, was used to modify reaction yields. These results indicated that GS-AFI was derived from reaction with NOF and that GS-AFII could be formed from both NOF and N-HO-AF. A reaction scheme is proposed in which NOF reacts with GSH to form an intermediate addition product that can rearrange either to GS-AFI or be reduced to GS-AFII. The latter could also be formed by direct reaction with N-HO-AF.

Amino Acids↗