Glucuronidation and excretion of nonylphenol in perfused rat liver.

Nonylphenol, an environmental estrogenic chemical, is reported to have adverse effects on the reproductive organs of animals. In this study, the metabolism of nonylphenol and that of other alkylphenols in the rat liver was investigated using liver perfusion. Alkylphenols (nonylphenol, hexylphenol, butylphenol, and ethylphenol) were glucuronidated by rat liver microsomes. Nonylphenol was found to be conjugated with glucuronic acid by an isoform of UDP-glucuronosyltransferase, UGT2B1, expressed in yeast AH22 cells. However, when nonylphenol was perfused into rat liver in situ, it was difficult for free nonylphenol and conjugated metabolite to be excreted into the bile or vein, and most of the perfused nonylphenol remained free and as a glucuronide conjugate in the liver tissue, even after 1 h of perfusion. After 1 h of perfusion of the other alkylphenols, most of them were excreted into the bile as glucuronides. Ethylphenol, which has the shortest alkyl chain, was excreted rapidly into both the bile and vein; however, the excretion rates of alkylphenols having longer alkyl chains tended to be slow. MRP-2-deficient Eisai hyperbilirubinemic rats could not secrete alkylphenol-glucuronides into the bile, indicating that alkylphenol-glucuronides are transported by MRP-2 to the bile in normal Sprague-Dawley rats. The results indicate that the kinetics of excretion of alkylphenol-glucuronides into the bile or vein depends on the length of alkyl chain and suggest that nonylphenol-glucuronide formed in the liver cannot be transported by MRP-2.     

Glucuronidation of 1-naphthol in the rat intestinal loop

The glucuronidation of 1-naphthol after its instillation into the intestinal lumen was studied in closed or single pass perfused jejunal loops by measuring the appearance of 1-naphthol glucuronide in the intestinal lumen and in venous blood. Within 30 min 69% of 1-naphthol instilled into the jejunal loop was metabolized almost exclusively to the glucuronide; 49% was in venous blood and 20% in the intestinal lumen. After instillation of 1-naphthol glucuronide, 59% was left in the intestinal lumen and 20% appeared in venous blood. When the intestine was perfused with 1-naphthol, 91% of the naphthol absorbed from the intestinal lumen was glucuronidated at a rate of 6.7 nmole/min/g tissue; 82% of the glucuronide was released into the blood under steady state conditions. The results demonstrate the suitability of this system for studies on drug metabolism in intact intestinal tissue.     

Flow-dependent extraction of 1-naphthol by the rat isolated perfused kidney

Summary The influence of variation of perfusion flow rate on the renal clearance of p-aminohippuric acid and 1-naphthol was studied with an isolated perfused rat kidney preparation. Kidney functions were well maintained at low perfusion flow rates by the use of a fluorocarbon emulsion to increase the oxygen capacity of the perfusion buffer. Renal extraction of p-aminohippuric acid decreased with increasing perfusion flow. Our data show that at high perfusion flow rates maximal extractable perfusion flow forms only a small part of the total perfusion flow. 1-Naphthol is rapidly metabolized to its glucuronide and sulfate conjugate in the isolated perfused rat kidney. Using PAH as a marker for the maximal extractable perfusion flow, 1-naphthol could be regarded as a high-extraction compound even at high perfusion flow rates. Our results suggest that p-aminohippuric acid clearance, rather than total perfusion flow rate, should be used as the measure of maximal extractable blood flow for the estimation of extraction ratio in the isolated perfused kidney of compounds excreted or metabolized by the proximal tubules.     

Prednisolone metabolism and excretion in the isolated perfused rat kidney

The isolated perfused rat kidney was used to identify factors responsible for the renal elimination of prednisolone (Pn). Pn was recirculated at initial concentrations varying from 100 to 1000 ng/ml for 90 min. Perfusate and urine samples were assayed for Pn and prednisone by HPLC. Protein binding of Pn was measured by using 3H-Pn and equilibrium dialysis at 37 degrees C. There were no significant differences in perfusate flow, glomerular filtration rate, urine flow, or sodium excretion between control and steroid experiments. Partial metabolism of Pn to prednisone occurred in all studies. The total kidney clearance (CIT) of Pn ranged from 0.39 to 1.24 ml/min/100 g of rat body weight with approximately half of the Pn dose unaccountable for as either Pn or prednisone. The apparent percentage of the Pn dose excreted unchanged in the urine ranged from 1.9 to 6.4% and was not related to Pn dose. The apparent urinary clearances of Pn and its metabolite, prednisone, normalized for inulin clearance (fractional excretion) were variable with means of 0.068 and 0.095, respectively. The fractional excretions of Pn and prednisone were related to the fraction of filtered water excreted but not to perfusate concentration. Thus, the extent of urinary clearance of these corticosteroids is related to glomerular filtration and passive tubular reabsorption. The perfused rat kidney reflects the urinary and renal metabolic clearance of Pn without the complication of dose-dependent disposition.     

Glucuronidation of 1-naphthol in nuclear and microsomal fractions of the human intestine

The glucuronyl transferase activity was measured with 1-naphthol as a substrate in nuclear and microsomal fractions of the human intestinal mucosa. The mucosa was obtained from different parts of the intestine. The rate of 1-naphthol glucuronidation ranged between 0.70 and 1.26 nmol/mg/min (nuclear fraction) and 0.21 and 0.54 nmol/mg/min (microsomal fraction). The average (+/- SEM) of the nuclear/microsomal ratios of the glucuronyl transferase was 2.48 +/- 0.19. The epoxide hydrolase activity towards styrene oxide was measured in the same subcellular fractions; it was undetectable in the nuclear fraction, whereas it was 0.46 +/- 0.04 nmol/mg/min (mean +/- SEM) in the microsomal fraction. The glucuronyl transferase activity was also measured in nuclear and microsomal fractions of the human liver. The activity (mean +/- SEM) was 1.14 +/- 0.21 nmol/mg/min (nuclei) and 5.00 +/- 0.80 (microsomes). The average of the nuclear to microsomal ratios (mean +/- SEM) was 0.32 +/- 0.03.     

Metabolism and excretion of S-conjugates derived from hexachlorobutadiene in the isolated perfused rat kidney

Renal processing of the S-conjugates derived from hexachlorobutadiene (HCBD), S-(pentachlorobutadienyl)glutathione (PCBG), and S-(pentachlorobutadienyl)-L-cysteine (PCBC) was studied in the isolated perfused rat kidney. At an initial perfusate concentration of 20 microM, both conjugates were rapidly eliminated from the perfusate. Calculation of the fractional clearance rates revealed the dominant role of nonfiltering mechanisms in this process. This was confirmed by the strong inhibitory effect of 50 microM probenecid. S-(Pentachlorobutadienyl)-N-acetyl-L-cysteine (N-Ac-PCBC) was detected as the major metabolite of both PCBG and PCBC in urine and perfusate. PCBC and S-(pentachlorobutadienyl)cysteinylglycine were minor urinary metabolites formed from PCBG; only N-Ac-PCBC and PCBC were detected in the perfusate. At an initial S-conjugate concentration of 100 microM in the perfusate, the rate of elimination of both PCBG and PCBC continuously decreased during the perfusion, mainly as the result of a reduced excretion of N-Ac-PCBC. This indicates marked disturbance of N-acetylation and/or transport under these conditions. Addition of probenecid resulted in a significantly reduced renal elimination of both S-conjugates, predominantly due to a reduced rate of mercapturate excretion. In contrast, the nephrotoxicity of PCBC or PCBG was not significantly influenced by probenecid. It is concluded from these experiments that the kidney has the capacity to metabolize HCBD S-conjugates and that nonfiltering excretion of the mercapturic acid plays a decisive role. The pathways of HCBD S-conjugate metabolism in the kidney were shown to be dependent on their initial concentrations in the perfusate, most probably as a consequence of concentration-dependent toxic disturbances of transport and/or N-acetylation.     

Absorption and presystemic glucuronidation of 1-naphthol in the vascularly fluorocarbon emulsion perfused rat small intestine

Summary Using the isolated vascularly fluorocarbon emulsion perfused rat small intestine some factors which determine the extent of the intestinal glucuronidation of 1-naphthol to 1-naphthol--d-glucuronide were studied. Increasing the luminal 1-naphthol concentration resulted in a concomitant increase in the 1-naphthol appearance in the vascular perfusate. In contrast, the total appearance of 1-naphthol--d-glucuronide increased less than proportional to the increase in the luminal 1-naphthol concentration. About 88% of the total amount of 1-naphthol--d-glucuronide excreted was released into the vascular perfusate. The capacity-limited intestinal glucuronide efflux is most likely due to saturation of the excretory mechanism for 1-naphthol--d-glucuronide. Decreasing the vascular flow rate influenced both the appearance of 1-naphthol and 1-naphtol--d-glucuronide in the vascular perfusate, whereas the appearance of 1-naphthol--d-glucuronide in the luminal perfusate was essentially flow-independent. A noradrenaline-induced change in the haemodynamic state of the vascular bed (with the total flow kept constant) resulted in a marked decrease in the 1-naphthol vascular concentration. The vascular 1-naphthol--d-glucuronide concentration was only slightly affected. These results indicate that changes in blood flow and blood flow distribution within the intestinal wall can affect the extent of presystemic intestinal metabolism by interfering with the absorption of the parent compound and the efflux of formed conjugates. These parameters can be of paramount importance for causing variable intestinal first-pass effects of drugs in vivo. Send offprint requests to M. H. de Vries at the above address     

Renal excretion mechanisms of the antiviral nucleoside analogue AM 188 in the rat isolated perfused kidney

1. AM 188 is an antiviral guanosine analogue that undergoes extensive renal excretion in humans. The present study was designed to investigate the disposition of AM 188 over a range of concentrations in the rat isolated perfused kidney (IPK) to explore the mechanisms involved in its renal handling. 2. Right kidneys of male Sprague-Dawley rats (n = 23) were isolated and perfused in recirculating mode with Krebs'-Henseleit (pH 7.4) buffer containing 0.65% bovine serum albumin, 3.6% dextran, amino acids and glucose. [14C]-Inulin was added to the perfusate reservoir to permit estimation of glomerular filtration rate (GFR). [3H]-AM 188 and unlabelled AM 188 were added to the perfusate as a bolus initially, followed by a constant rate of infusion at 5, 25, 125, 500 or 1000 microg/min to achieve initial target perfusate concentrations of 1, 5, 25, 100 or 200 microg/mL, respectively. During the 130 min over which AM 188 was infused, urine was collected in 10 min intervals (commencing 10 min after the bolus dose) and perfusate was collected at the mid-point of these intervals to permit calculation of the renal clearance (CLR) of AM 188. Binding of AM 188 in perfusate, measured using ultrafiltration, was negligible. 3. The bolus dose and infusion regimen produced relatively stable AM 188 concentrations in perfusate in the 5, 25 and 125 micro g/min groups and progressively increasing concentrations in the 500 and 1000 microg/min groups. High-pressure liquid chromatography analysis of IPK perfusate and urine suggested that there was no or negligible metabolism of AM 188 in the kidney. The CLR/GFR ratio for AM 188 (mean+/-SD) was 5.76 +/- 1.57, 5.99 +/- 0.52, 6.02 +/- 1.47, 3.38 +/- 0.26 and 1.08 +/- 0.42 in the 5, 25, 125, 500 and 1000 microg/min groups, respectively, showing significant reductions at the two highest infusion rates (P < 0.05). Although there was no difference between the five groups in the distribution of AM 188 between kidney tissue and perfusate (KT/P), at the end of perfusion the corresponding urine-to-tissue concentration ratio declined significantly in the 1000 microg/min group. 4. AM 188 undergoes substantial net renal secretion over a wide range of perfusate concentrations. A reduction in renal clearance at perfusate concentrations above 25 microg/mL could be due to saturation of carrier-mediated transport at the brush border membrane and/or a solubility limitation leading to precipitation of AM 188 in tubular cells and/or tubular urine.     

Taurocholate excretion and bile formation in the isolated perfused rat liver

Summary The excretory transport maximum (Tm) for taurocholate was determined in the perfused rat liver and compared to that obtained in the intact rat. An in situ liver perfusion system employing a semisynthetic perfusion medium containing Krebs-Ringer-bicarbonate solution, bovine erythrocytes and bovine albumin was used.In contrast to the bromosulfophthalein (BSP)-Tm reported previously, the taurocholate-Tm was 45% smaller in vitro (196±17 nmol/min per g liver) than in vivo (357±10 nmol/min per g liver), indicating that bile salt transport is more susceptible to alterations induced by the conditions of the perfusion than BSP transport. These findings add to the differences observed previously between the hepatic handling of anionic dyes and bile salts.At a low taurocholate infusion rat (57 nmol/min per g liver) the normal relationship between bile salt excretion and bile flow observed in vivo was maintained in the perfused liver. At higher taurocholate infusion rates, however, bile flow, for a given bile salt excretion rate, was smaller than in vivo.These findings should be taken into account when the isolated perfused rat liver is employed for studies of bile formation.     

UDP-glucuronyltransferase in perfused rat liver and in microsomes—III. Effects of galactosamine and carbon tetrachloride on the glucuronidation of 1-naphthol and bilirubin

Galactosamine treatment (400 mg/kg, i.p., 4 hr) markedly decreased the level of UDP-glucuronic acid (UDPGA) and 1-naphthol glucuronidation in perfused liver. In contrast, bilirubin glucuronidation was not affected. In non-activated microsomes both 1-naphthol and bilirubin glucuronidation were dependent upon the concentration of UDPGA. In UDP-N-acetylglucosamine-activated microsomes, 1-naphthol glucuronidation remained dependent upon UDPGA whereas bilirubin glucuronidation tended to be independent of UDPGA.Carbon tetrachloride treatment (5 ml/kg, per os, 24 hr) strongly decreased 1-naphthol glucuronidation in the intact liver without altering the level of UDPGA. Bilirubin glucuronidation was affected similarly but to a lesser extent, In contrast. 1-naphthol glucuronidation in liver microsomes was increased under these conditions. In the presence of UDP-N-acetylglucosamine and UDP, however, enzyme activity in microsomes from CCl₄-treated rats was lower than in control microsomes.The results suggest a differential regulation of 1-naphthol and bilirubin glucuronidation and stress the importance of intracellular effectors for glucuronidation in the intact liver.     

Vasodilator effects of dopaminomimetics in the perfused rat kidney

The renal vascular effects of dopaminomimetics were studied in the isolated perfused rat kidney after pretreatment with 10(-5) M phenoxybenzamine and 10(-5) M sotalol and after contraction of the vascular bed with prostaglandin F2 alpha (10(-7) to 3 x 10(-6) M). Under these conditions, our criterion for vascular dopaminomimetic activity was renal vasodilation, competitively inhibited by d-butaclamol (10(-8) M) but not by 1-butaclamol (3 x 10(-8) M). Dopamine is active at micromolar concentrations (ED50 = 2.53 +/- 0.34 x 10(-6) M). The N-alkylated analogues of dopamine preserve this activity if the alkyl group is a methyl group (epinine) or two n-propyl groups (di-n-propyl-dopamine). The catechol nucleus appears essential for renal vascular dopaminomimetic activity (SK&F 38393, active; p-tyramine, di-n-propyl-m-tyramine and RU 24926, inactive). Bromocriptine reproduces renal dopaminergic vasodilation with an ED50 of 1.3 +/- 0.14 x 10(-6) M. The study of structure-activity relationships of dopaminomimetics relates the vascular dopamine receptor, associated with renal vasodilation, to the dopamine receptor, associated with stimulation of dopamine-sensitive adenylate cyclase.     

Glomerular filtration and saturable absorption of iohexol in the rat isolated perfused kidney.

1. The renal handling of iohexol was examined in the rat isolated perfused kidney (IPK) over a perfusate concentration range of 5-20 micrograms ml-1. 2. At a concentration of 5 micrograms ml-1, a ratio of renal clearance over clearance by glomerular filtration (ClR/GF) of 0.63 +/- 0.06 could be determined. This ratio increased until 1.02 +/- 0.06 at 20 micrograms ml-1, indicating that a saturable mechanism is involved in the luminal disappearance of the drug. 3. Pretreatment of the kidneys with polylysine, probenecid or diatrizoate resulted in a significantly enhanced clearance of iohexol, probably due to inhibition of membrane binding. Renal clearance data were fitted to a kinetic model including filtration into the primary urine followed by saturable absorption at the luminal membrane. An absorption constant, KA, of 7.3 +/- 1.3 micrograms ml-1, and a maximum rate of absorption, VA,Max, of 1.4 +/- 0.1 micrograms min-1 were determined. 4. Iohexol accumulated in kidney tissue, reaching a concentration of 2 to 7.5 times the perfusate concentration. In freshly isolated proximal tubular cells and kidney cortex mitochondria, iohexol reduced the uncoupled respiratory rate at a concentration comparable to the highest tissue concentration found in the IPK. 5. In conclusion, iohexol is not only filtered by the kidney but also reabsorbed via a saturable mechanism, which results in tubular accumulation. Intracellularly sequestered iohexol may affect mitochondrial oxidative metabolism. Our results indicate that iohexol is not a true filtration marker.     

Biosynthesis and biliary excretion of S-conjugates of hexachlorobuta-1,3-diene in the perfused rat liver.

The formation and biliary excretion of the glutathione and cysteine S-conjugates of hexachlorobutadiene were studied in the isolated perfused rat liver. Infusion of increasing amounts of hexachlorobutadiene led to an increase in total metabolite excretion. Partitioning of glutathione conjugate release between bile and perfusate depended on the rate of substrate infusion: S-(1,2,3,4,4-pentachlorobutadienyl)glutathione (PCBG) appeared quantitatively in bile at low hexachlorobutadiene infusion rates, and increasing amounts of the glutathione conjugate were found in the perfusate as infusion rates were increased. The cysteine S-conjugate, S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine, was not detected in the perfusate, but the amounts found in the bile were correlated with the concentrations of the glutathione conjugate. Depletion of hepatic glutathione concentrations decreased PCBG formation. Hence, at moderate hexachlorobutadiene infusion rates, PCBG is exclusively excreted into bile, indicating that intestinal absorption of PCBG or its metabolites is required for the induction of kidney damage in vivo.     

Malaria infection impairs glucuronidation and biliary excretion by the isolated perfused rat liver.

1. The effect of the erythrocyte stage of malaria infection on hepatic glucuronidation, biliary excretion and oxidation processes was investigated using harmol, salbutamol, taurocholate and propranolol. Livers from rats infected with the rodent malaria parasite P. berghei were isolated and perfused in a single-pass (harmol, taurocholate, propranolol) or recirculating (harmol, salbutamol) design. The degree of erythrocytic parasitaemia ranged from 16% to 63%. 2. The hepatic clearance (Cl) of harmol decreased from 7.8 +/- 0.4 ml/min in controls to 5.7 +/- 1.1 ml/min in the malaria-infected group in single-pass studies. This corresponded to a 40-60% reduction in hepatic intrinsic clearance (Clint). Similar changes were observed using the recirculating design when glucuronidation accounted for greater than 90% of harmol metabolism. 3. The Cl of salbutamol, metabolized exclusively by glucuronidation under the conditions used, also decreased significantly from 8.5 +/- 0.8 in controls to 6.6 +/- 1.4 ml/min in the malaria-infected group. This corresponded to a 40-70% reduction in Clint. 4. The Cl of taurocholate, excreted unchanged in bile, decreased slightly but significantly from 9.6 +/- 0.3 ml/min in controls to 8.3 +/- 0.9 ml/min in the malaria-infected group. In the same livers, there was also a slight but significant decrease in propranolol Cl (10.0 +/- 0.1 ml/min and 9.9 +/- 0.1 ml/min, respectively). Both these compounds undergo flow-limited hepatic clearance; the decreases in Clint of taurocholate and propranolol were 87% and 35%, respectively. 5. Cl and Clint of each of the compounds studied were found to correlate significantly with the degree of erythrocytic parasitaemia. This study shows that glucuronidation, biliary excretion and oxidation by liver are impaired in malaria infection in rats, with biliary excretion being the most affected. The data indicate that there is a general decrease in hepatic elimination processes during the erythrocytic phase of malaria infection.     

Disposition of quinapril and quinaprilat in the isolated perfused rat kidney

An isolated perfused rat kidney model was used to probe the renal disposition of quinapril and quinaprilat after separate administration of each drug species. Control studies were performed with drug-free perfusate (n=8) and perfusate containing quinapril (n=9) quinaprilat (n=7) at initial drug concentrations of 1000 ng/ml (including corresponding tracer levels of tritiated drug). Physiologic parameters were within the normal range of values for this technique and were stable for the duration of each experiment. Quinapril and quinaprilat concentrations were determined in perfusate, urine, and perfusate ultrafiltrate using a specific and sensitive reversed-phase HPLC procedure with radiochemical detection, coupled to liquid scintillation spectrometry. Perfusate protein binding was determined using an ultrafiltration method at 37°C. The total renal learance of quinapril (CLr) was calculated asDose/AUC (0-∞), and is represented by the sum of its urinary and metabolic clearances. The urinary clearances (CLe) of quinapril and quinaprilat were calculated as urinary excretion rate divided by midpoint perfusate concentration for each respective species. Of the total renal clearance for quinapril (CLr=4.49 ml/min), less than 0.1% was cleared as unchanged drug (CLe=0.004 ml/min); over 99% of the drug was cleared as quinaprilat formed in the kidney. The clearance ratio of quinapril [CR=CLr/(fu·GFR)] was 41.0, a value representing extensive tubular secretion into the renal cells. Following quinaprilat administration, the clearance ratio of metabolite [CR=CLe/(fu β GFR)] was 3.85, indicating a net secretion process for renal elimination. This work was supported in part by a gift from Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company and by Grant R01 GM35498 from the National Institutes of Health.     

Conjugation of 1-naphthol and transport of 1-naphthol-conjugates in the vascularly perfused small intestine of the mouse

A method is described which allows the simultaneous vascular and luminal perfusion of the murine small intestine. This preparation was used for the investigation of 1-naphthol conjugation in the gut and the sidedness of conjugate release. The viability of this preparation can be maintained for more than 1 hr as indicated by morphological controls, measurement of tissue metabolism and the transport of 3-O-methyl-glucose against a concentration gradient. When 100 μM 1-naphthol was administered on the luminal side, it was conjugated at a constant rate, yielding 1-naphthyl-glucuronide and 1-naphthyl-sulfate in a molar ratio of 1:2. Both metabolites were excreted into the blood at the contraluminal side of the epithelium. The results are discussed with respect to the sidedness of intestinal transport systems for anionic conjugates of xenobiotics and drugs.     

The use of urinary N-acetyl-beta-glucosaminidase in human renal toxicology. I. Partial biochemical characterization and excretion in humans and release from the isolated perfused rat kidney.

Selected biochemical properties and the human excretory pattern of urinary N-acetyl-β-glucosaminidase were characterized. A renal origin of the urinary enzyme was demonstrated by comparison of the release from the isolated perfused rat kidney with excretion from the in situ organ. The rate of activity excretion from both the perfused and in situ organ was 22% of the total organ content per day. The basal enzyme release was into tubular luman only, and contralumenal release into the renal lymphatic system was essentially zero. The human urinary activity exhibited unusual properties which indicated that the rate of excretion is directly proportional to the corresponding rate of release by the tubular epithelium. In urine, 0.2% of the activity is lost per hour at 37°C, and the activity is not denatured by physiologic variations in urine pH and osmolarity. The urinary activity is not associated with cells, is almost entirely soluble and exhibits no latency upon addition of Triton X-100. A cortico-medullary activity gradient exists in the human kidney. The rate of activity excretion in 30 human subjects was constant under maximal variations in urine flow rate. The 3-day excretion in 21 subjects fluctuated around a fairly constant mean rate, which was characteristic of each individual, and subsequent 3-day collections up to 3 weeks later were identical. The rate of excretion was independent of body mass, lean body mass, and sex. The pattern of N-acetyl-β-glucosaminidase excretion fluctuated identically with that of several other acid hydrolases. It is concluded that, with certain restrictions, N-acetyl-β-glucosaminidase excretion can be employed as a marker of the rate of release of at least several acid hydrolases from the human kidney.