Rat Liver Preparations Research Articles

The chemistry and biology of 7H-dibenzo[c,g]carbazole: synthesis and characterization of selected derivatives, metabolism in rat liver preparations and mutagenesis mediated by cultured rat hepatocytes.

Authentic stable standards of 7H-dibenzo[c,g]carbazole (DBC), a potent environmental carcinogen, were synthesized in order to study the compound's metabolism and mutagenesis in whole cell systems. Complete characterization of 2-OH-DBC, 3-OH-DBC, 4-OH-DBC, 13c-OH-DBC and N-methyl-DBC was accomplished by UV, IR, fluorescence and high resolution NMR spectra, and by high resolution mass spectrometric procedures. Metabolites of DBC were isolated and separated by HPLC from extracts of rat liver microsomal incubations and the medium of primary cultures of rat liver cells. Identification of metabolites was accomplished by comparisons between the authentic standards and isolated metabolites by UV and fluorescence spectroscopy, mass spectral analyses, and by co-chromatographic techniques. 2-OH-DBC and 3-OH-DBC were found in all rat liver preparations as well as three other unidentified phenols. 4-OH-DBC, 13c-OH-DBC or N-methyl-DBC were not isolated under any conditions. The rates of appearance of DBC metabolites in cultures of rat liver cells were compared to those for benzo[a]pyrene (BaP) at 10, 25, 50 and 100 microM substrate. At 25 microM substrate or greater, DBC metabolites appeared in the culture medium at significantly faster rates than those of BaP. At 100 microM substrate, DBC metabolites appeared at a rate approximately 4-times the rate observed for BaP. When the mutagenic potential of DBC was compared to that of BaP under identical conditions in a co-cultivation system of rat liver cells and an epithelial cell line, DBC was found to produce significantly higher rates of mutagenesis than BaP at concentrations of 0.4, 4.0 and 40.0 microM in the culture medium. The mutagenic potential of DBC was compared to that of several derivatives of the parent compound. 3-OH-DBC, 13c-OH-DBC and N-methyl-DBC were found to be mutagenic in the co-cultivation system at 40 microM, with mutation frequencies of 4.4 +/- 0.8, 8.0 +/- 3.1 and 12.9 +/- 5.4 mutants per 10(5) survivors, respectively. The parent compound induced 8.0 +/- 2.8 mutants per 10(5) survivors at the same concentration. 2-OH-DBC and 4-OH-DBC were not mutagenic under the same conditions. The studies have shown that metabolism of 7H-DBC leads predominantly to phenols in rat liver cells. The results of the mutagenesis experiments indicate that, of the derivatives studied, those associated by induction to the nitrogen are mutagenic. The latter studies suggest that the nitrogen is involved in the activation of the parent compound through inductive mechanisms.

Hydroxylation of phenylethylamine by rat liver preparations. Inhibition studies

1. 1. Rat liver 100,000 g pellet microsomal fraction p-hydroxylate phenylethylamine to tyramine in a relatively slow proceeding, NADPH-requiring reaction; K m 2.1 × 10 −5 M and V max 0.32 nmol/mg protein/20 min. 2. 2. This reaction is inhibited, either competitively, noncompetitively or uncompetitively by a number of behaviorally active monomethylated and monohalogenated derivatives of phenylethylamine. 3. 3. Whereas formation of tyramine was not significantly affected by l-phenylalanine or its p-chloro derivative, it was competitively inhibited by imipramine, iprindole and alprazolam. 4. 4. It is suggested that at least some of the effects of these drugs may result from their ability to interfere with phenylethylamine metabolism.

N-oxygenation of stobadine, a gamma-carboline antiarrhythmic and cardioprotective agent: the role of flavin-containing monooxygenase.

1. N-oxygenation of stobadine, a gamma-carboline antiarrhythmic and cardioprotective agent, was investigated in vitro using rat liver preparations. 2. Stobadine N-oxygenase activity was located mainly in the microsomal fraction and exhibited a requirement for oxygen and NADPH. The apparent Km and Vmax values for the process were, respectively, 350 microM and 3.48 nmol/mg protein per min. 3. N-oxygenation of stobadine in rat liver microsomes was not affected by SKF 525-A, carbon monoxide, metyrapone, cyanide or n-octylamine. When guinea-pig liver microsomes were used the reaction was activated by n-octylamine. 4. N-oxygenase activity was strongly inhibited by methimazole and depressed by phenobarbital and 3-methylcholantrene pretreatment. 5. The above results, along with the pH optimum of 8.4, strongly indicate the involvement of flavin-containing monooxygenase in the metabolic N-oxygenation of stobadine. 6. Species difference in liver microsomal N-oxygenase activity was evident, the order of activity being guinea-pig greater than rat greater than rabbit.

Zinc Protoporphyrin Inhibits CO Production in Rats

We studied the effect of zinc protoporphyrin (ZnPP) on in vivo total bilirubin production, as measured by the excretion rate of carbon monoxide (VeCO), in adult rats. A single subcutaneous dose of ZnPP (40 μmol/kg) suppressed the VeCO between 2–12 h posttreatment, with maximum suppression of approximately 20% by 4 h. The heme oxygenase activity in the liver and spleen of the ZnPP‐treated rats was significantly inhibited at 12 h compared with that of the controls. The in vitro inhibition of heme oxygenase activity by the addition of exogenous ZnPP to native rat liver and spleen tissue preparations was observed, as evidenced by suppressed CO production. This in vitro inhibition of heme oxygenase activity by ZnPP was further confirmed by finding decreased bilirubin formation, as measured directly by high‐performance liquid chromatography. This study demonstrates that ZnPP can inhibit in vivo liver and spleen heme oxygenase activity and suppress CO production in the rat, as well as inhibit in vitro heme oxygenase activity in native rat tissues.

Zinc protoporphyrin inhibits CO production in rats.

We studied the effect of zinc protoporphyrin (ZnPP) on in vivo total bilirubin production, as measured by the excretion rate of carbon monoxide (VeCO), in adult rats. A single subcutaneous dose of ZnPP (40 mumol/kg) suppressed the VeCO between 2-12 h posttreatment, with maximum suppression of approximately 20% by 4 h. The heme oxygenase activity in the liver and spleen of the ZnPP-treated rats was significantly inhibited at 12 h compared with that of the controls. The in vitro inhibition of heme oxygenase activity by the addition of exogenous ZnPP to native rat liver and spleen tissue preparations was observed, as evidenced by suppressed CO production. This in vitro inhibition of heme oxygenase activity by ZnPP was further confirmed by finding decreased bilirubin formation, as measured directly by high-performance liquid chromatography. This study demonstrates that ZnPP can inhibit in vivo liver and spleen heme oxygenase activity and suppress CO production in the rat, as well as inhibit in vitro heme oxygenase activity in native rat tissues.

Mutagenicity testing of rutacridone epoxide and rutacridone, alkaloids in Ruta graveolens L., using the Salmonella/microsome assay.

The mutagenicity of rutacridone and rutacridone epoxide was investigated using Salmonella typhimurium without as well as with different metabolic activation systems. Rutacridone epoxide was found to be a direct acting mutagen in S. typhimurium strains TA98, TA100 and TA1538; addition of rat liver preparations resulted in a marked decrease of mutagenicity. In contrast, rutacridone required metabolic conversion to exhibit mutagenic activity. Neither of the compounds had any effect on tester strain TA1978. S9 mixes as well as microsomal and cytosolic preparations from untreated, phenobarbital-treated and 3-methylcholanthrene-treated rats were used to study the activation and deactivation capacities of the enzyme mixtures. In addition, the influence of enzyme inhibitors on the activation and deactivation of the furoacridones were tested. Evidence is presented that rutacridone is metabolized by rat liver enzymes to the corresponding epoxide as the ultimate mutagen.

The Toxicokinetics of (1R,Cis)- and (1R,Trans)-Tetramethrin in the Isolated Perfused Rat Liver

1. The toxicokinetics of cis- and trans-tetramethrin isomers were investigated using the isolated perfused rat liver preparation. 2. The concentration of cis- and trans-tetramethrin decreased rapidly in the plasma perfusate and was initially replaced by N-(hydroxymethyl)3,4,5,6-tetrahydrophthalimide (MTI) and then by 3,4,5,6-tetrahydrophthalimide (TPI). Plasma perfusate concentrations of the intact cis-isomer were higher than those of the trans-isomer. Concentrations of MTI and TPI were higher in livers treated with the trans-isomer. 3. Tetramethrin and its metabolites were rapidly excreted in the bile. Bile from livers perfused with trans-isomer contained higher concentrations of parent isomer and metabolites MTI and TPI, than did bile from livers treated with the cis-isomer.

Effects of perfusate flow rate on measured blood volume, disse space, intracellular water space, and drug extraction in the perfused rat liver preparation: characterization by the multiple indicator dilution technique.

The effect of hepatic blood flow on the elimination of several highly cleared substrates was studied in the once-through perfused rat liver preparation. A constant and low input concentration of ethanol (2.0 mM), [14C]-phenacetin and [3H]-acetaminophen (0.36 and 0.14 microM, respectively), or meperidine (8.1 microM) was delivered once-through the rat liver preparation in five flow periods (greater than 35 min each); control flow periods at 12 ml/min were interrupted by flow changes to 8 or 16 ml/min. The steady-state hepatic availabilities (F or outflow survivals) at 12 ml/min were ethanol, 0.075 +/- 0.038; [14C]-phenacetin, 0.15 +/- 0.059; [3H]-acetaminophen, 0.34 +/- 0.051; meperidine, 0.047 +/- 0.017. Flow-induced changes were different among the compounds: with reduced flow (8 ml/min), F was decreased for ethanol (0.061 +/- 0.032) and [3H]-acetaminophen (0.28 +/- 0.051), as expected, but was increased for [14C]-phenacetin (0.20 +/- 0.068) and meperidine (0.05 +/- 0.03); with an elevation of flow (to 16 ml/min), F was increased for all compounds, as expected of shorter sojourn times: ethanol, 0.13 +/- 0.065; [14C]-phenacetin, 0.22 +/- 0.062; [3H]-acetaminophen, 0.43 +/- 0.063; meperidine, 0.055 +/- 0.022. A marked increase in F for ethanol had occurred when flow changed from 12 to 16 ml/min due to nonlinear metabolism; the latter was confirmed by a reduction in the extraction ratios at increasing concentrations (1.8 to 11.4 mM); this condition was not present for the other compounds. In order to explain the observations, we used the multiple indicator dilution technique to investigate the flow-induced behaviors of tissue distribution spaces of vascular and intracellular references in the perfused rat liver preparation.

Competing pathways in drug metabolism. II. An identical, anterior enzymic distribution for 2- and 5-sulfoconjugation and a posterior localization for 5-glucuronidation of gentisamide in the rat liver.

Gentisamide (2,5-dihydroxybenzamide, GAM), a substrate that forms two monosulfates at the 2 and 5 positions (GAM-2S and GAM-5S) and a monoglucuronide at the 5 position (GAM-5G), was delivered at 8 or 80 microM by normal (N) and retrograde (R) flows to the once-through rat liver preparation. At the lower (8 microM) input concentration, ratios of conjugate formation rate, GAM-5S/GAM-5G and GAM-2S/GAM-5G, were decreased significantly (4.01 +/- 1.42 to 2.93 +/- 0.99, and 1.13 +/- 0.65 to 0.66 +/- 0.41, respectively) whereas a small but significant increase in the steady-state extraction ratio, E (0.89 +/- 0.029 to 0.94 +/- 0.016), was observed upon changing the flow direction from N to R. At the higher input GAM concentration (80 microM), conjugate formation rate ratios were relatively constant for GAM-5S/GAM-5G (1.18 +/- 0.08 to 1.11 +/- 0.12) and GAM-2S/GAM-5G (0.33 +/- 0.05 to 0.31 +/- 0.05) upon changing flow direction from N to R, despite a slight increase in E from 0.87 +/- 0.023 to 0.91 +/- 0.016 was observed. These results suggest that the sulfotransferase activities responsible for 2- and 5-sulfoconjugations are identically distributed and localized anterior to 5-glucuronidation activities during a normal flow of substrate into the rat liver (entering the portal vein and exiting the hepatic vein), and that the presence of uneven distribution of conjugation activities is discriminated only at the lower input drug concentration. At high concentration (greater than Km for all systems), saturation of all pathways occurs, and other anteriorly/identically distributed competing pathways would fail to perturb downstream intrahepatic drug concentrations and the resultant conjugation rates. The lack of change in metabolic profiles renders the condition unsuitable for examination of uneven distribution of enzymes in the liver. These observations were generally predicted by theoretical enzymic models of consistent distribution patterns. Because 2- and 5-sulfation were mediated by systems of similar Km but different Vmax values, two possibilities, the same isozyme of sulfotransferase being involved in the formation of two enzyme-substrate complexes to form two distinctly different products or two isozymes of sulfotransferases of identical distribution, were discussed.

Rapid metabolic inactivation of tipredane, a structurally novel topical steroid

[ 3H]Tipredane ([ 3H]TP), [ 3H]triamcinolone acetonide ([ 3H]TAAC), [ 3H]hydrocortisone ([ 3H]HC), and [ 3H]betamethasone-17α-valerate ([ 3H]BMV), each at a concentration of 1 μM, were separately incubated with the 10,000 g supernatant fraction of the liver and skin homogenates of humans, rats and mice (BMV was studied only in human liver). Sequential samples were taken for up to 1 h during each incubation. The radioactivity in each sample was extracted with methanol, and the methanolic extracts were analyzed by high performance liquid chromatography. Among the four compounds studied, [ 3H]TP was most rapidly biotransformed by the liver preparations of the three species. The rates of in vitro biotransformation of TP were 2.5–30 times faster than those of TAAC, HC and BMV. In the human liver preparation, the rates of biotransformation were in the order of: TP > TAAC > HC = BMV. In the mouse and rat liver preparations, the orders were: TP > TAAC > HC and TP > HC > TAAC, respectively. In the skin preparations, little, if any, biotransformation of [ 3H]TP and [ 3H]TAAC was observed in any of the species studied; however, [ 3H]HC underwent a slow, steady biotransformation in the skin preparations of humans and rats but not of mice. [ 3H]TP was biotransformed by the liver preparations of all three species to numerous metabolites, thirteen of which have been identified. The biotransformation reactions included: (1) sulfoxidation; (2) elimination of either one or both alkylthio groups; and (3) hydroxylation of the steroid nucleus. Some metabolites were synthesized and tested for glucocorticoid receptor binding and anti-inflammatory activities; all were found to be much less potent than TP. The observed separation of local anti-inflammatory activity from systemic side effects of TP is most probably due to its rapid metabolic inactivation; the liver, rather than the skin, is mainly responsible for the metabolic inactivation of TP.

Identification of LH/hCG receptors in rabbit uterus.

Luteinizing hormone (LH) is believed to act via specific receptors to control gonadal steroidogenesis and reproductive processes. Recently A. J. Ziecik, P. D. Stanchev, and J. E. Tilton (Endocrinology 119:1159, 1986) reported surprisingly that LH/hCG receptors were present in porcine uterus, a tissue not known to be a target for LH action. We report herein the identification of high-affinity LH receptors in the rabbit uterus. Uteri from adult New Zealand white rabbits were homogenized in Tris-HCl, 0.25 M sucrose. After filtration and sequential centrifugation, a partially purified pellet containing receptors was obtained. This preparation was incubated with a trace (1300 cpm) (50 pg) 125I-labeled chorionic gonadotropin and with various unlabeled protein hormones. Receptor bound was separated from free hormone by centrifugation at 1000 g. Affinity was estimated by Woolf plot analysis. Specific binding sites for LH/hCG were identified. The following Kd's were calculated: human LH, 1.6 X 10(-11); hCG, 0.5 X 10(-11); human TSH, 1.3 X 10(-9); and human FSH, 7.85 X 10(-9). The reaction of human FSH and TSH with the receptor is best explained by LH contamination of these hormones. A similar preparation of rat liver showed that no binding sites were present. Rabbit ovarian LH receptors had a Kd slightly higher at 4.1 X 10(-11) than that of the uterine LH receptors. Rabbit ovarian receptors were present at 2.27 X 10(-13) M/mg protein compared to uterine receptors at 4.65 X 10(-15) M/mg protein. We conclude specific- and high-affinity binding sites (receptors) for LH are present in the rabbit uterus. The function of these receptors remains unknown.

Uptake and Excretion of Vecuronium Bromide and Pancuronium Bromide in the Isolated Perfused Rat Liver

Using the isolated perfused rat liver preparation, the disappearance from the perfusate and the excretion in the bile of vecuronium bromide and pancuronium bromide and their metabolites were followed for 2 h after the addition of 1 mg of either drug to the perfusate. In addition, the rate of change of the hepatic content of these two compounds was calculated by serially subtracting the amount of the compound and the metabolites in the bile and in the perfusate from the dose of drug added to the perfusate. It was found that, whereas the concentration of pancuronium in the perfusate declined slowly and monoexponentially, vercuronium concentration in the perfusate declined rapidly in a biexponential manner. No metabolites of either drug were detected in the perfusate. Approximately 40% of the injected dose of vecuronium was excreted in the bile as unchanged vecuronium and another 30% as the 3-hydroxy metabolite. No other metabolites of vecuronium were found in the bile. In total only about 7% of pancuronium (unchanged) was collected in the bile by the end of the experiment. It is concluded that, in comparison to pancuronium, the rat liver takes up large amounts of vecuronium rapidly, half of which is eliminated as unchanged vecuronium and half as the 3-hydroxy derivative. A small amount of vecuronium or its 3-hydroxy metabolite is returned to the perfusate from the liver. Some possible mechanisms underlying these differences are discussed.

Stereochemistry of the major rodent liver microsomal metabolites of thecarcinogen dibenz[a,j]acridine.

The major metabolites of the carcinogen dibenz[a,j]acridine formed in rodent liver microsomal preparations were trans-3,4-dihydroxy-3,4-dihydrodibenz[a,j]acridine (DBAJAC-3,4-DHD) and dibenz[a,j]acridine 5,6-oxide (DBAJAC 5,6-oxide) [Gill et al. (1987) Carcinogenesis 8, 425-431]. The enantiomers of DBAJAC-3,4-DHD were prepared from the separable diastereoisomeric esters with (+)-endo-1,4,5,6,7,7-hexachlorobicyclo[2.2.1]hept-5-ene-2-carboxyl ic acid (HCA). The absolute configuration of trans-3(R),4(R)-dihydroxy-1,2,3,4-tetrahydrodibenz[a,j]acridine was assigned by conversion to the bis[p-(dimethylamino)benzoate] and examination of the exciton coupling in its circular dichroic (CD) spectrum. The 3(R),4(R)-tetrahydrodiol was converted to DBAJAC-3(R),4(R)-DHD. The enantiomers of DBAJAC 5,6-oxide were partially resolved by chiral stationary-phase chromatography, and subsequent methoxide attack afforded two enantiomerically enriched isomeric ethers from each fraction. The structures of the two ethers from each enantiomer were determined, and from their 1H NMR spin-spin coupling between the H5 and H6 signals and the CD spectra of the ethers, the absolute configuration of the ethers, and hence the 5,6-oxides, was determined. The enantiomeric composition of the 3,4-dihydrodiol and 5,6-oxide formed as microsomal metabolites of rat liver preparations was 69% 3R,4R and 81% 5R,6S, respectively. When rats were pretreated with 3-methylcholanthrene (MC), these percentages were 70% and 5%, indicating a reversed stereochemical preference for oxide formation in the MC-induced preparation. Results are also presented for phenobarbitone-induced rat liver and mouse liver preparations.

Lack of linear correlation between hepatic ligand uptake rate and unbound ligand concentration does not necessarily imply receptor-mediated uptake

Previous studies of the hepatic uptake of several albumin-bound ligands, using constant and variable albumin concentrations, were interpreted as being inconsistent with the traditional mechanism of uptake, defined as an uptake rate directly proportional to unbound ligand concentration, and led to the formulation of the albumin receptor theory of hepatic uptake. Because other experimental designs have failed to confirm the albumin receptor theory, we reexamined, using the isolated perfused rat liver preparation, the traditional uptake mechanism under the conditions used in the original studies, of constant and variable albumin concentration. Livers (n = 6) were perfused in randomized sequence with 10 different solutions containing 24-14C-taurocholate in a single-pass design. Five solutions contained fixed albumin (0.1 mM) and variable taurocholate (3-48 microM) concentrations, and five maintained the taurocholate-albumin ratio fixed at 0.06; absolute concentrations of taurocholate varied from 3-48 microM, and of albumin from 0.05-0.08 mM. At constant albumin concentration in hepatic inflow, elimination rate of taurocholate was linearly related to both total (Cin) and unbound (Cin,u) taurocholate concentration in hepatic inflow, indicating first-order elimination kinetics. When taurocholate and albumin were increased in hepatic inflow in a fixed molar ratio, taurocholate uptake rate was not linearly related to Cin,u but was still consistent with the traditional uptake mechanism. Moreover, the apparent saturation of taurocholate uptake by added albumin was consistent with the reduction in unbound fraction (fu) in accordance with the traditional uptake mechanism. This study shows that although the traditional uptake mechanism dictates that ligand uptake rate is linearly related to unbound ligand concentration within the liver, uptake rate need not necessarily be linearly related to Cin,u. Therefore, experiments in which lack of a linear relationship between uptake rate and Cin,u is found do not necessarily imply receptor-mediated uptake.

Lactogenic and somatogenic binding sites in intact and detergent-solubilized membrane preparations of female rat liver.

The presence of lactogenic and somatogenic binding sites in intact microsomal membranes and in detergent-solubilized microsomal membrane preparations of female rat liver has been studied by affinity cross-linking-SDS/polyacrylamide-gel electrophoresis. In microsomal membrane preparations an Mr 40,000 lactogenic binder is present which is not disulphide-linked to another protein. Triton X-100 solubilization of membranes results in the appearance of three lactogenic 125I-human growth hormone (125I-hGH) binders with Mr values of 87,000, 40,000 and 35,000, and one somatogenic 125I-hGH binder with Mr 32,000. Treatment of rats with oestrogen increased the amount of lactogenic and somatogenic binding species in liver. The lactogenic binding sites are present as one entity in Triton X-100-solubilized preparations, clearly separated from the somatogenic binder as analysed by gel chromatography. Furthermore, 125I-hGH interacts with an Mr 95,000 somatogenic binder in membrane preparations to which the hormone can be cross-linked only following Triton X-100 solubilization.

Inhibitors of cholesterol biosynthesis. Further studies of the metabolism of 5α-cholest-8(14)-en-3β-ol-15-one in rat liver preparations

5α-Cholest-8(14)-en-3β-ol-15-one is a potent inhibitor of sterol biosynthesis in mammalian cells in culture and has significant hypocholesterolemic activity upon oral administration to rodents and non-human primates. The conversion of the 15-ketosterol to cholesterol upon incubation with the 10,000 × g supernatant fraction of rat liver homogenate preparations under aerobic conditions has been reported (D.J. Monger, E.J. Parish and G.J. Schroepfer, Jr. (1980) J. Biol. Chem. 255, 11122–11129). Presented herein are results of studies of the metabolism of [2,4- 3H]5α-cholest-8(14)-en-3β-ol-15-one obtained upon incubation with the microsomal, cytosolic and the 10,000 × g supernatant fractions of liver homogenates of female rats under a variety of conditions. The results of these studies indicated metabolism of the 15-ketosterol to materials with the chromatographic properties of fatty acid esters of the 15-ketosterol, fatty acid esters of C 27-monohydroxysterols, a component similar to the 15-ketosterol (possibly an isomer of the Δ 8(14)-15-ketosterol), and a polar component. Detailed studies of the C 27-monohydroxysterols obtained from incubation of the 15-ketosterol under anaerobic conditions indicated the formation of labeled 5α-cholesta-8, 14-dien-3β-ol and a 5α-cholest-7-en-3β-ol which were characterized by their behavior on silicic acid column chromatography, by the behavior of their acetate derivatives on medium pressure liquid chromatography on alumina-AgNO 3 columns, and by co-crystallization of the labeled sterols with authentic unlabeled standards. The identification of 5α-cholesta-8, 14-dien-3β-ol and 5α-cholest-7-en-3β-ol as metabolites of the 15-ketesterol, coupled with previous studies of the metabolism of 5α-cholesta-8, 14-dien-3β-ol and of 5α-cholest-8(14)-ene-3β, 15α-diol and 5α-cholest-8(14)-ene-3β, 15β-diol has permitted the formulation of a scheme for the overall metabolism of the 15-ketosterol to cholesterol.

Hepatic metabolism of vasoactive intestinal polypeptide (VIP) in the rat

Vasoactive intestinal polypeptide (VIP) is released into the portal circulation by a meal stimulus, but is rapidly cleared from plasma. Although it is known to bind to receptors on liver cells, the role of the liver in the clearance of VIP is not clearly defined. We therefore studied the disappearance of VIP in recirculating and in single pass isolated perfused rat liver (IPRL) preparations. Disappearance of added VIP was rapid in recirculating IPRL experiments with a half life of ca. 30 min. In single-pass steady-state studies in which livers were perfused at 16 ml/min for 30 min, clearance of VIP was complete (16 ml/min) at concentrations of 500 fmol/ml, but clearance fell to 3 and 1 ml/min at perfusate concentrations of 8 and 40 pmol/ml respectively. Further experiments to evaluate whether VIP was disappearing in perfusate itself demonstrated substantial metabolism of VIP in perfusate which had previously been circulated through a liver for 90 min. The products of metabolism were identical to those found in the IPRL. We conclude that VIP is rapidly cleared as it passes through the isolated perfused rat liver model with a significant proportion of clearance attributable to release of a peptidase from the liver into the perfusate.

Intestinal first-pass metabolism of nitrosamines. 2. Metabolism of N-nitrosodibutylamine in isolated perfused rat small intestinal segments.

In a previous study a high first-pass metabolism of N-nitro-sodi-[1-14C]butylamine (NDBA, 0.32-730 microM) has been shown in isolated perfused rat small intestinal segments. In the present work the identification and quantitation of metabolites in samples of perfusate and absorbed fluid (absorbate) is reported. After HPLC enrichment and purification five metabolites could be identified by GLC-MS: N-nitrosobutyl-(3-hydroxybutyl)amine (NB3HBA), N-nitrosobutyl-(4-hydroxybutyl)amine (NB4HBA), N-nitrosobutyl-(3-carboxypropyl)amine (NB3CPA), N-nitrosobutyl-(2-hydroxy-3-carboxypropyl)amine (NB2H3CPA) and N-nitrosobutylcarboxymethylamine (NBCMA), representing greater than 95% of total metabolites. omega-hydroxylation leading to NB4HBA and NB3CPA accounted for 75-95% of total metabolites. The formation of their follow-up products NB2H3CPA and NBCMA was too small for quantitative analysis. In absorbate NB3CPA was by far the most important metabolite. The hydroxylation of NB4HBA to NB3CPA was more efficient in jejunal as compared to ileal segments. omega-1-hydroxylation to NB3HBA and, as reported previously, alpha-hydroxylation were only minor metabolic pathways. In conclusion, a high first-pass metabolism of NDBA to the proximate bladder carcinogen NB3CPA takes place during its absorption in rat small intestine. This is in contrast to the observation in rat liver preparations, where alpha- and omega-1-hydroxylation are the predominant pathways. The intestinal first-pass metabolism may play a key role explaining the increased incidence of bladder tumors in rats after low oral doses of NDBA.

Disposition of the antimalarial, mefloquine, in the isolated perfused rat liver

The disposition of mefloquine has been investigated in the isolated perfused rat liver (IPRL) preparation after the administration of [ 14C]mefloquine HCl (3.8 mg, 4 μCi, quinoline ring labeled). Mefloquine underwent avid hepatic uptake within 10 min of dosing. Also at this point, hepatic oxygen consumption was reduced markedly in four of the six IPRL preparations, but was restored completely by approximately 30 min post-dose. The drug concentration profile underwent a biexponential decline over the 4-hr study period, with a terminal T 1 2 of 1.0 ± 0.3 hr. The area under the perfusate plasma concentration/time curve (AUC 0-∞) was 4.0 ± 1.8 μg·hr·ml −1. Mefloquine was a high clearance compound (956.0 ± 390 ml/hr) with a large apparent volume of distribution (1416 ± 819 ml) in the IPRL. Biliary excretion accounted for 7.5 ± 6.5% of the dose. Mefloquine was quantitated by HPLC analysis as approximately half (3.3 ± 1.8%) of biliary label, the remainder consisting of highly polar metabolites of mefloquine. By 4 hr, a total of 64.8 ± 4.4% of the [ 14C] dose was recovered from the livers. Subsequent HPLC analysis revealed this to be mostly unchanged mefloquine. Subcellular fractionation of the homogenized livers revealed that 50.6 ± 6.8% of the dose of mefloquine was located in the 10,000 g pellet. In summary, mefloquine was cleared rapidly from the IPRL and underwent avid hepatic uptake into the lipid-rich fractions of rat liver.

Mutagenicity of furoquinoline alkaloids in the Salmonella/microsome assay. Mutagenicity of dictamnine is modified by various enzyme inducers and inhibitors.

Furoquinoline alkaloids are activated to mutagens by microsomal preparations of rat liver. The mutagenic effects decrease with the increasing number of methoxyl-substituents on the furoquinoline skeleton. After metabolic activation dictamnine, gamma-fagarine and skimmianine exhibit strong mutagenicity in Salmonella typhimurium strains TA98 and TA100 and have comparatively little or no activity in the corresponding non-R-factor strains TA1538 and TA1535. This indicates that these compounds primarily act as frameshift mutagens. The activation capacity of the metabolizing mixture depends on the amount of microsomal protein. Pretreatment of male Wistar rats with phenobarbital (Pb) or 3-methylcholanthrene results in an increase in the metabolic capacity of the corresponding liver microsome preparations, Pb induction showing the greater effect. Various enzyme inhibitors, such as carbon monoxide, metyrapone, SKF-525A, 7,8-benzoflavone and methimazole, decrease the activation capacities of rat liver preparations, whereas 1,1,1-trichloropropene-2,3-oxide has no effect. These results suggest that furoquinolines are activated to mutagenic metabolites by cytochrome-P-450 and cytochrome-P-448, and possibly by the flavin-containing monooxygenase.