TY - JOUR
AU - Csanády, György, A.
AB - Abstract The industrial chemical 1,3-butadiene (BD) is a potent carcinogen in mice and a weak one in rats. This difference is generally related to species-specific burdens by the metabolites 1,2-epoxy-3-butene (EB), 1,2:3,4-diepoxybutane (DEB), and 3,4-epoxy-1,2-butanediol (EBD), which are all formed in the liver. Only limited data exist on BD metabolism in the rodent liver. Therefore, metabolism of BD, its epoxides, and the intermediate 3-butene-1,2-diol (B-diol) was studied in once-through perfused livers of male B6C3F1 mice and Sprague-Dawley rats. In BD perfusions, predominantly EB and B-diol were found (both species). DEB and EBD were additionally detected in mouse livers. Metabolism of BD showed saturation kinetics (both species). In EB perfusions, B-diol, EBD, and DEB were formed with B-diol being the major metabolite. Net formation of DEB was larger in mouse than in rat livers. In both species, hepatic clearance (ClH) of EB was slightly smaller than the perfusion flow. In DEB perfusions, EBD was formed as a major metabolite. ClH of DEB was 61% (mouse) and 73% (rat) of the perfusion flow. In the B-diol–perfused rat liver, EBD was formed as a minor metabolite. ClH of B-diol was 53% (mouse) and 34% (rat) of the perfusion flow. In EBD-perfused rat livers, ClH of EBD represented only 22% of the perfusion flow. There is evidence for qualitative species differences with regard to the enzymes involved in BD metabolism. The first quantitative findings in whole livers showing intrahepatic first-pass metabolism of BD and EB metabolites will improve the risk estimation of BD. 1,3-butadiene, 1,2-epoxy-3-butene, 3-butene-1,2-diol, 3,4-epoxy-1,2-butanediol, 1,2:3,4-diepoxybutane, mouse and rat liver The gaseous olefin 1,3-butadiene (BD) is a major industrial chemical used primarily in the production of synthetic rubbers and of plastics such as copolymers of BD with styrene, with acrylonitrile and styrene, and with methyl methacrylate and styrene. It is also an intermediate in the synthesis of basic petrochemicals (IARC, 1999). In long-term (6 h/day, 5 days/week, 2 years) carcinogenicity studies, inhaled BD was weakly tumorigenic in rats exposed to 1000 or 8000 ppm (Owen et al., 1987) but was highly effective in mice exposed to BD concentrations ranging from 6.25 to 625 ppm. In females, tumors increased at a concentration as low as 6.25 ppm in the lungs and at 20 ppm in various sites. In male mice, the lowest effective BD concentration was 62.5 ppm showing increased tumor incidences in several organs. In both genders, increased tumor incidences were found in every investigated tissue at 200 ppm (Melnick et al., 1990). In order to understand the cause for this species difference, metabolism of BD was intensively investigated in several laboratories. A series of metabolic intermediates has been detected in exhaled air, blood, and urine (summarized in USEPA, 2002). From genotoxicity studies (reviewed in, e.g., IARC, 1999; USEPA, 2002), it was concluded (Fred et al., 2008) that the tumorigenicty of BD resulted primarily from the burden and the relative genotoxic potency of the three mutagenic epoxide metabolites 1,2-epoxy-3-butene (EB), 1,2:3,4-diepoxybutane (DEB), and 3,4-epoxy-1,2-butanediol (EBD). The latter can be formed by hydrolysis of DEB or by oxidation of 3-butene-1,2-diol (B-diol), the hydrolytic product of EB (Fig. 1). All four have been found in BD-exposed rats and mice (reviewed in Filser et al., 2007). Oxidative metabolism of BD, EB, and B-diol is catalyzed by cytochrome P450 (CYP)–dependent monooxygenases as was shown with liver microsomes (BD: Bolt et al., 1983; Cheng and Ruth, 1993; Csanády et al., 1992; Duescher and Elfarra, 1994; Elfarra et al., 1991; Filser et al., 1992; Malvoisin et al., 1979a; Sharer et al., 1992; Wistuba et al., 1989; EB: Csanády et al., 1992; Krause and Elfarra 1997; Kreuzer et al., 1991; Malvoisin et al., 1979b; Malvoisin and Roberfroid, 1982; Seaton et al., 1995; B-diol: Kemper et al., 1997, 1998; Krause et al., 2001). Endoplasmic epoxide hydrolase (EH) catalyses the hydrolysis of EB to B-diol (Csanády et al., 1992; Krause et al., 1997; Kreuzer et al., 1991; Malvoisin and Roberfroid, 1982; Malvoisin et al., 1981). The hydrolysis of DEB was postulated to result in EBD and that of EBD in 1,2,3,4-tetrahydroxybutane. The latter product was shown in DEB containing microsomal incubations prepared from livers of mice, rats, and humans (Boogaard and Bond, 1996). From this picture, concurrent interactions of BD and its metabolites at the metabolizing enzymes had been postulated (Henderson, 2001). Earlier findings showed in BD-exposed rats much less EB than expected from the amounts of BD metabolized (Filser and Bolt, 1984). Information on a first-pass metabolism of intrahepatically produced BD metabolites like EB can be obtained when using the perfused liver as was shown in a preliminary report on BD metabolism at very high BD concentrations (Filser et al., 2001). In addition, a meaningful use of this system can be expected to deliver more reliable values of the kinetic parameters of BD and its epoxides than subcellular liver fractions which are far from the in vivo situation. Trustworthy values of these kinetic parameters are of utmost importance, when using in a physiological toxicokinetic model for risk estimation purposes. Consequently, the goal of the present work was to study quantitatively the metabolism of BD, EB, DEB, B-diol, and EBD in once-through perfused livers of mice and rats. FIG. 1. Open in new tabDownload slide Investigated metabolic pathways of BD and its metabolites. For simplification, the optical isomers of the BD metabolites are not shown. FIG. 1. Open in new tabDownload slide Investigated metabolic pathways of BD and its metabolites. For simplification, the optical isomers of the BD metabolites are not shown. MATERIALS AND METHODS Chemicals All commercial chemicals were purchased with the highest purity available from Riedel-de-Haën (Seelze, Germany) and Merck (Darmstadt, Germany). Liquemin N25000 (Heparin-Natrium) was from Hoffmann La Roche (Grenzach-Wyhlen, Germany), bovine serum albumin (fraction V) from Roche Diagnostics (Mannheim, Germany), and isoflurane (Isoba vet) from Essex Tierarznei (München, Germany). BD 99.5% was obtained from Linde (München, Germany). EB 98%, racemic DEB 97%, 1,2-epoxybutane 99%, n-butylboronic acid 97%, 4-benzylpiperidine 99%, and Krebs-Henseleit buffer powder were from Sigma-Aldrich (Steinheim, Germany). Several compounds were obtained by custom synthesis: racemic B-diol 98+% from EMKA-Chemie (Neufahrn, Germany) and EBD 98% (meso:racemic = 1:4), perdeuterated 1,2:3,4-diepoxybutane (DEB-D6) 98% (meso:racemic = 1:1), perdeuterated 3-butene-1,2-diol (B-diol-D8) 98%, and 3,4-epoxy-[1,1,2,3,4,4-2H6]-butane-1,2-diol (EBD-D6) 98% (meso:racemic = 1:2) from Synthon (Augsburg, Germany). Handling of all chemicals during different sample preparations was carried out under the hood. Animals Male Sprague-Dawley rats (200–300 g) and male B6C3F1 mice (17–25 g) were purchased from Charles River Wiga GmbH (Sulzfeld, Germany). All experimental procedures with animals were performed in conformity with the Guide for the Care and Use of Laboratory Animals (NRC, 1996) under the surveillance of the authorized representative for animal welfare of the Helmholtz Zentrum München. Up to 1 week before use, two rats or five mice each were housed in the Institute of Toxicology in a Macrolon type III cage placed in an IVC top flow system (Tecniplast, Buguggiate, Italy). This system provided the animals with filtered room air. A constant 12-h light/dark cycle was maintained in the chamber room. Animals had free access to standard chow (Nr. 1324 from Altromin, Lage, Germany) and tap water. Liver Perfusion Erythrocytes were prepared from bovine blood, freshly collected in a slaughterhouse. The fresh blood was immediately added to Liquemin N25000 (5000 IU heparin/ml), resulting in a heparin concentration of 30 IU/ml blood. The erythrocytes gained after centrifuging (2000 × g; 11 min; room temperature) were washed three times in Krebs-Henseleit buffer (pH 7.4) and then stored at 4°C (not longer then 24 h) until use for the production of the perfusate required for the perfusion experiments. The perfusate consisted of Krebs-Henseleit buffer (pH 7.4) containing 1% (wt/vol) of albumin and 40% (vol/vol) of bovine erythrocytes. It was oxygenated by room air (see below). After deeply anesthetizing an animal by isoflurane inhalation, the abdominal cavity was opened. Rat livers were connected to the 37°C-warm flow of oxygenated perfusate via the vena portae and then isolated as described in detail in Sies (1978). The vena cava inferior served as outlet for the effluent perfusate at the right atrium. Mouse livers were treated correspondingly with the exception that they were not isolated from the carcass in contrast to rat livers (see below). On the basis of the liver perfusion technique described by Miller et al. (1951), a gastight perfusion system (Fig. 2) was developed enabling liver perfusions with constant concentrations of volatile compounds. Perfusions were carried out once through. A glass beaker (3 l) warmed to 37°C in a water bath served as perfusion reservoir. A magnetic stirrer slowly but continuously stirred the perfusate, which resulted in an oxygen-hemoglobin saturation of 85–100%. A peristaltic pump transported the perfusate via a silicone tube, which was conducted in several coils through a 37°C-warm water bath, to the all-glass 12-bulb oxygenator. The oxygenator, too, was warmed by a continuous flow of water to 37°C. From here, a second peristaltic pump equipped with Tygon tubing carried the perfusate predominantly in Teflon tubes (also warmed up to 37°C) through a Teflon-coated rubber septum of the all-glass liver chamber to the indwelling catheter in the vena portae of the liver. Both peristaltic pumps were adjusted for perfusion flows (Q) of about 20 ml/min (rat livers) or 4 ml/min (mouse livers). Before entering the liver, the perfusate passed a gas trap made out of glass and a Teflon-coated rubber septum (situated immediately in front of the gastight liver chamber). The latter enabled to collect perfusate samples for the analysis of BD, its metabolites, pH, and blood gases. The perfusate leaving the liver via the indwelling catheter in the vena cava inferior at the right atrium flowed through a flexible Tygon tube which left the liver chamber through a Teflon-coated rubber septum. A septum mounted into the hose enabled to take samples of the effluent perfusate, immediately behind the exposure chamber. FIG. 2. Open in new tabDownload slide System for once-through perfusions of volatile or gaseous substances through rat or mouse livers (for detailed description see text). a, magnetic stirrer; b, perfusate reservoir; c, peristaltic pump; d, oxygenator; e, gas bubble trap; f, closed all-glass rat liver chamber; g, closed all-glass mouse liver chamber; h, thermometer; i, septum for collecting influent perfusate samples; j, septum for collecting effluent perfusate samples; k, all-glass sphere (64 l); l, magnetic stirring motor and magnetic propeller; m, vacuum gauge; n, membrane pump; o, Teflon piston pump; p, silicon tube containing flowing water (37°C). FIG. 2. Open in new tabDownload slide System for once-through perfusions of volatile or gaseous substances through rat or mouse livers (for detailed description see text). a, magnetic stirrer; b, perfusate reservoir; c, peristaltic pump; d, oxygenator; e, gas bubble trap; f, closed all-glass rat liver chamber; g, closed all-glass mouse liver chamber; h, thermometer; i, septum for collecting influent perfusate samples; j, septum for collecting effluent perfusate samples; k, all-glass sphere (64 l); l, magnetic stirring motor and magnetic propeller; m, vacuum gauge; n, membrane pump; o, Teflon piston pump; p, silicon tube containing flowing water (37°C). Two all-glass chambers were used, one for rat and one for mouse liver perfusions. The rat liver perfusion chamber consisted of a glass cylinder with a dome-shaped bottom and a gastight attachable dome-shaped glass lid. The bottom was equipped with a closable escape and two laterally mounted Teflon-coated rubber septa. One served as outlet for the perfusate and the other one to collect gas probes from the chamber atmosphere for control purposes. The lid was closed upward by two Teflon-coated rubber septa. The upper one served as inlet for the perfusate, the more lateral one as socket for an alcohol thermometer. The whole chamber except the lid was surrounded by a series of coils of a silicon tube which was run through by water of 53°C, thus warming the air of the chamber (256 ml) to exactly 37°C as was controlled by the thermometer. The glass lid could be removed in order to allow placing the liver into the chamber. The liver was hanging freely from the tightly fixed catheter in the vena portae. The structure of the mouse liver chamber was very similar to that of the rat liver chamber with the exception that it was much larger (1100 ml), allowing the placement of the operation table on which the liver of the anesthetized mouse was prepared for perfusion. Because of its higher fragility when compared to the rat liver, the mouse liver was perfused in situ with only the lower part of the body being surgically removed. The chamber air was warmed up to 37°C by a water-perfused Silicon hose that was twined around both the lower part of the chamber and its lid. The lid contained a septum that was opened when closing the lid. In order to enrich the perfusate with the gaseous BD, a gas administration system was developed which is also outlined in Figure 2. An all-glass sphere (64 l) mounted on a magnetic stirring motor was equipped with a magnetic propeller lying on its bottom and with an 8-cm long glass neck (inner diameter 15 cm). It was covered by a round glass lid containing four Teflon-coated rubber septa. One of them served as closable connection to a Teflon piston pump by which a pressure reduction to 300 Torr could be achieved. Two others were linked to the airspace of the oxygenator via Teflon hoses and a membrane pump. The Teflon hoses were provided with two glass stopcocks. Before starting a BD-perfusion experiment, a vacuum was established in the all-glass sphere. Then, the amount of BD gas required for a certain concentration in the perfusate was injected by means of a disposable syringe via the fourth septum. Thereafter, the septum was shortly opened to allow fast pressure equalization with fresh room air. Through the same septum, the BD concentration in the atmosphere of the sphere was monitored during the perfusion experiment by repeatedly collecting air samples for gas chromatographic (GC) analysis. After supplying a cannulated liver with oxygenated perfusate and inserting the liver into the chamber, the perfusion was started immediately; the stopcocks were opened allowing the BD-air mixture to maintain a closed circuit between the sphere and the 12-bulb oxygenator (air space 1 l), and the membrane pump was started (air flow: 2.4 l/min). The air stream was in countercurrent flow with the downward perfusate flow. By this method, a continuous and uniform distribution of BD in the perfusate was achieved. For the administration of all the nongaseous liquid compounds EB, DEB, B-diol, or EBD into the influent perfusate, a different system was used. In order to study the metabolism of each of these BD daughters, a defined amount of the corresponding substance was diluted with Krebs-Henseleit buffer to establish the desired concentration in the perfusate. The obtained solution was filled into a gastight glass infusion syringe (100 ml, series 1000; Hamilton, Bonaduz, Switzerland). By means of an infusion pump (Precidor type 5003; Infors, Basel, Switzerland), the solution was pumped (1 ml/min for rat livers and between 0.2 and 0.6 ml/min for mouse livers) into the flowing perfusate via a Teflon-coated rubber septum that was mounted in the Teflon tubing which transported the perfusate from the gas trap to the liver chamber. Behind this septum, at a distance of about 30 cm from the septum used for monitoring the influent perfusate, a mixing chamber was installed in the Teflon tubing. The mixing chamber consisted of a glass tube (6 cm × 15 mm) with three inblown indentations at its rear side (toward the liver). It contained an about 4-cm long, on both ends sealed, glass tube that left a small cylindrical slot between the inner wall of the outer tube and the outer wall of the inner tube. The perfusate was considerably accelerated when flowing through this slot and was effectively swirled by the indentations before arriving well mixed at the liver chamber. In these perfusion experiments, the oxygenator was opened to the environmental air. The whole perfusion system was arranged in a fume hood. During each perfusion, perfusate samples of 1 ml were collected repeatedly in front of and behind the liver, and the partial pressures of oxygen and CO2 as well as the pH (adjusted to 7.4) were repeatedly monitored using a blood gas analyzer (pHOx; Nova Biomedical, Waltham, MA). In front of the livers, the oxygen partial pressure varied from 50 to 135 mm Hg (oxygen-hemoglobin saturation 85–100%, Thews et al., 1991) and the CO2 partial pressure from 23 to 43 mm Hg. Behind the livers, the corresponding values were between 36 and 120 mm Hg (oxygen-hemoglobin saturation 70–100%) and between 23 and 42 mm Hg, respectively. The values are in reasonable agreement with partial pressures of O2 and CO2 measured in rat livers in vivo (summarized by Tolboom et al., 2007). Within a perfusion experiment, the partial pressures of both gases in front of or behind the livers varied usually not more than 10%. The effluent flows measured repeatedly in each experiment were 17–21 ml/min (rat) and 3.7–4.5 ml/min (mouse). Corresponding in vivo reference values are 20.8 and 4.25 ml/min (Arms and Travis, 1988). The biochemical integrity of the livers was verified by measuring the concentration ratio lactate-to-pyruvate as a measure of the redox state of the liver (Williamson et al., 1967). The concentrations of lactate and pyruvate in the efflux of the liver were determined photometrically using diagnostic kits (no. 826 and 726) obtained from Sigma-Diagnostics (St Louis, MA). From the lactate-to-pyruvate ratios that were between 15 and 30, the liver conditions were judged as good to moderate (according to Oomen and Chamalaun, 1971). Perfusion experiments were maintained up to 72 min. Perfusion experiments. Mouse livers were once-through perfused with BD, EB, DEB, or B-diol and rat livers with the same substances and in addition with EBD, in order to obtain quantitative information on the fate of each of these compounds during its first liver passage. In agreement with the metabolic scheme given in Figure 1, influent and effluent perfusates were analyzed for BD, EB, B-diol, DEB, and EBD in perfusion experiments with BD, for EB, DEB, B-diol, and EBD in perfusion experiments with EB, for DEB and EBD in DEB perfusions, for B-diol and EBD when perfusing with B-diol, and only for EBD in rat liver perfusion experiments with EBD. Analytical Methods In the following, the analytical methods are described that were used in the perfusion experiments with BD, EB, and B-diol. 1,3-Butadiene. BD in the perfusate was determined by a headspace method. An aliquot (1 ml) of the perfusate was injected into a gastight headspace vial (7.5–7.9 ml). After determining the exact volume of the perfusate sample by weighting, the vial was incubated in a shaking water bath for at least 20 min at 37°C in order to achieve equilibrium between the gas and liquid phase. Gas samples (25 μl) were taken from the headspace and injected on column in a Shimadzu GC-8A gas chromatograph (GC) equipped with a flame ionization detector. Separation was done on a stainless steel column (3.5 m × 1/8” × 2 mm) packed with Tenax TA (60–80 mesh; Chrompack, Frankfurt, Germany) using N2 as carrier gas. The limit of detection (three times the background noise) was 0.9 ppm in the atmosphere of the headspace vial. The equilibrium concentration measured in the gas phase together with the partition coefficient perfusate-to-air (0.63 at 37°C; Bhowmik, 2002) was used to calculate the actual BD concentration in the perfusate as the sum of BD in the gas phase and in the liquid phase of the headspace vial. 1,2-Epoxy-3-butene. A sample of 1 ml was taken from the perfusate using a 1-ml disposable syringe that contained 10 μl of an ethanolic solution of diethyl maleate (1 mol/l; in order to deplete glutathione [GSH] in the erythrocytes) and immediately injected into a closed headspace vial (2 ml) together with 10 μl of an ethanolic solution of the internal standard 1,2-epoxybutane. After thoroughly shaking, the whole sample was collected from the closed vial using a 1-ml disposable syringe. (For pressure compensation, a second needle was placed in parallel through the septum of the headspace vial.) The sample was directly transferred into a 1.5-ml Eppendorf cup containing 200 μl of dichloromethane, and the cup was closed instantaneously. After vortexing and centrifugation, the cup was cooled to 0°C on ice and 100 μl of the organic phase (lower layer) were collected by a Hamilton syringe and transferred into an injection bottle containing a glass insert (0.1 ml). The injection bottle was closed immediately with a crimp cap (silicon septum). After injecting 3 μl onto column, analysis was carried out with the GC HP 6890 equipped with the mass selective detector (MSD) HP 5973 (Agilent Technologies, Waldbronn, Germany) running in the positive chemical ionization mode with methane as reagent gas. Separation was done on a Zebron ZB-5–fused silica capillary column (30 m × 0.25 mm ID, film thickness 1.0 μm) or, alternatively, on an HP-5MS capillary column (30 m × 0.25 mm × 1.0 μm). A precolumn (fused silica capillary, deactivated) protected each of both columns. EB and 1,2-epoxybutane were quantified in the single ion monitoring (SIM) mode using the ions m/z = 71 for EB and m/z = 55 for 1,2-epoxybutane. The detection limit, defined as a signal to noise ratio of 3, was 0.01 μmol/l perfusate. 1,2:3,4-Diepoxybutane. The sample preparation for DEB was similar to that described for EB. An aliquot (1 ml) taken from the perfusate by means of a 1-ml syringe that contained 10 μl of the diethyl maleate solution was injected into a 2-ml Eppendorf cup and 10 μl of the ethanolic solution of the internal standard DEB-D6 was added. After shaking and adding 200 μl dichloromethane, the mixture was vortexed followed by centrifugation at 0°C. Then, about 100 μl of the lower dichloromethane layer was transferred into a glass insert (0.1 ml) in an injection bottle. The bottle was closed immediately and then stored at −80°C for not longer than a few days. For analysis, 4 μl of this solution was injected into the above-described GC/MSD that was running in the positive chemical ionization mode with ammonia as reagent gas. Separation was carried out on the same columns that were used for EB determination. Ions of 104 (DEB) and 110 (DEB-D6) were monitored in the SIM mode for quantification. Racemic DEB was separated from the meso-isomer. However, “DEB” in the “Results” section represents the sum of both diastereomers. The detection limit (three times the background noise) was 0.01 μmol/l perfusate for both the meso- and the racemic isomer. 3-Butene-1,2-diol and 3,4-epoxy-1,2-butanediol. B-diol and EBD were determined by GC/MSD after derivatization to cyclic n-butylboronic esters. Aliquots of the perfusate (6 ml each) were taken using disposable syringes containing 60 μl of an ethanolic diethyl maleate solution (1 mol/l) and, together with acetonic solutions of the corresponding internal standards (B-diol-D8 or EBD-D6), injected into screw-capped glass centrifugation vials (6 ml) that were immediately closed before removing the erythrocytes by centrifugation. The supernatants were stored at −80°C. On the day of B-diol analysis, 250 μl of the corresponding supernatant was derivatized with n-butylboronic acid (250 μl of a 0.04 molar acetonic solution) at room temperature. Then, 500-μl ethyl acetate was added, the mixture was centrifuged, and the upper organic layer containing the n-butylboronic ester was transferred into an autosampler vial followed by GC/MSD analysis. For EBD analysis, 3 ml of the EBD-D6 containing erythrocyte-free supernatant was mixed with 30 ml of freshly prepared i-propanol/chloroform (90/10, vol/vol) and thoroughly shaken. After adding potassium carbonate (3 g), thoroughly shaking again, and centrifuging, the upper organic layer was evaporated to dryness under a stream of nitrogen at 45°C. The residue was treated at room temperature with 600 μl of an acetonic solution of n-butylboronic acid (0.04 mol/l) and vortexed. Five minutes later, 500 μl of ethyl acetate was added and the mixture was centrifuged. The supernatant containing the n-butylboronic ester was transferred into an autosampler vial of the GC/MSD. Of the ethyl acetate extracts, 4 μl each was injected on column into the GC/MSD equipped with the same columns as described for EB. Detection was done in the positive ionization mode using methane (B-diol) or ammonia (EBD) as reagent gas. In the SIM mode, ions of 109 (B-diol), 161 (B-diol-D8), 188 (EBD), and 194 (EBD-D6) were monitored for quantitative analysis. Although both diastereomers of EBD were separated, their sum was used to quantify EBD. The detection limit (three times the background noise) was 0.02 μmol/l of perfusate for B-diol and for each of both EBD diastereomers. EBD was analyzed by this method in the BD perfusion experiments. The GC analytical methods presented above for BD and its metabolites are explicitly described in Bhowmik (2002). In perfusion experiments carried out with EBD and DEB, both substances were analyzed using liquid chromatography with tandem mass spectrometry (LC/MS/MS). 3,4-Epoxy-1,2-butanediol. The extraction from perfusate and the evaporation of the organic layer containing EBD and its internal standard EBD-D6 were carried out as described above (sample preparation for GS/MSD analysis). Then, both substances were derivatized to the corresponding 4-(4′-benzylpiperidin-1′-yl)-butane-1,2,3-triol by solving the residue in 500 μl of a methanolic solution of 4-benzylpiperidine (2 mg/ml) for 1 h at room temperature. After the addition of 250 μl acetonitrile, the samples were stored for 30 min on ice and then centrifuged. An aliquot of the supernatant (5 μl) was analyzed by LC/MS/MSD for the EBD derivatives. The analytical procedure is described in detail in Filser et al. (2007). The detection limit (three times the background noise) was 0.01 μmol/l when collecting perfusate samples of 6 ml. 1,2:3,4-Diepoxybutane. The sample preparation was done as described above for the GC determination of DEB with the exception that 800 μl dichloromethane were added to the perfusate aliquot of 1 ml. After centrifugation, the upper (aqueous) phase was carefully removed. Of the residual organic phase, 700 μl was given to a centrifugation vial and concentrated to near dryness under a gentle stream of nitrogen at 0°C in ice. Then, 50 μl of a methanolic solution of 4-benzylpiperidine (2 mg/ml) was added, the vial was closed, vortexed, and subsequently incubated in a water bath at 50°C for 5 h. Thereafter, the sample containing the 4-benzylpiperidine derivatives of DEB and its internal standard DEB-D6 was transferred into an autosampler vial of which 5 μl was subjected to LC/MS/MS analysis. The LC/MS/MS system consisted of an HP1100 liquid chromatograph (from Agilent Technologies) and an API 4000 triple quadrupole mass spectrometer with turbo ion spray interface (from Applied Biosystems, Darmstadt, Germany). The liquid chromatograph was equipped with a Luna C18 (2) column (150 × 2 mm id, 5 μm) obtained from Phenomenex (Aschaffenburg, Germany). Separation was carried out within 3.5 min at 24°C with a flow of 300 μl/min using an isocratic mobile phase of 5 mmol/l aqueous ammonium acetate (adjusted to pH = 4 with acetic acid) and methanol (50:50, vol/vol). The turbo ion spray source of the API 4000 was operated at a temperature of 700°C in the positive ionization mode at an ion spray voltage of 5400 V. Nitrogen served as curtain (CUR = 10), nebulizing (GS1 = 65, GS2 = 63), and collision gas (CAD = 12). The mass spectrometer was used in the multiple reaction–monitoring mode. Unit resolution was used for both Q1 and Q3. For quantification, the peak area of the transition ion at m/z 262 -> 116 (dwell time 50 ms, declustering potential = 110 V, collision energy = 47 V) was monitored for the DEB-derivative relative to that at m/z 268 -> 117 (dwell time 50 ms, declustering potential = 107 V, collision energy = 48 V) monitored for the DEB-D6 derivative. Additional fragmentation reactions were used as qualifiers. The not-separated diastereomers were eluted after a retention time of about 3 min. Data processing was done by means of the software Analyst 1.4.1 from Applied Biosystems. The detection limit of DEB (three times the background noise) was 0.005 μmol/l. Determination of the toxicokinetic parameters. Rate of metabolic elimination (dNel/dt) of a parent compound or net formation rate of a daughter metabolite (dNfi/dt) were calculated by multiplying the actual perfusion flow (pf; ml/h) with the difference between the mean concentrations in the influent (cin) and the effluent (cef) perfusate of the individual parent or daughter at steady state in a perfusion experiment; (dNel or fi/dt = pf·|cin − cef|). Net formation (fi%) of a daughter metabolite was related to the metabolic elimination of its metabolic precursor by dividing the net formation rate of the daughter (dNfi/dt) by the rate of metabolism of its parent (dNel/dt) and multiplying the result with 100; (fi% = 100·dNfi/dt/dNel/dt). In agreement with Wilkinson and Shand (1975), hepatic clearance (ClH) and intrinsic clearance (ClIn) of a compound were obtained by dividing an actual rate of metabolism by the mean concentration in the influent (ClH = dNel/dt/cin) and in the effluent (ClIn = dNel/dt/cef) perfusate, respectively. Clearance values were normalized for a body weight of a reference mouse and a reference rat of 25 and 250 g, respectively, by dividing the experimental values by the body weight of the rodent from which the liver was isolated and by multiplying the result with body weight of the corresponding reference animal. By the same procedure, actual rates of metabolic BD elimination were normalized for body weight. In order to obtain the maximum rate of BD metabolism (Vmax) and the apparent Michaelis constant (Kmap), kinetics according to Michelis and Menten were fitted to data points giving normalized rates of metabolic BD elimination versus the measured corresponding mean BD concentrations in the effluent perfusate. For fitting purposes, the program Prism 5 for Mac OS X was used (from GraphPad Software, La Jolla, CA). Means and SDs were calculated by the same program. RESULTS Typical concentration-time courses of BD and its metabolites detected in influent and effluent perfusate of BD-perfused livers are depicted in Figure 3A for mice and in Figure 3B for rats at a BD concentration in the effluent perfusate of 8.2 μmol/l (mice) and 20 μmol/l (rats). The dashed lines represent the means of a data set measured in the influent or effluent perfusate. BD, B-diol, and EB were detected in the effluent perfusate of all BD exposures. FIG. 3. Open in new tabDownload slide Typical concentration-time courses of BD and its metabolites EB, B-diol, and DEB in influent and effluent perfusate of a BD-perfused mouse (A) and rat (B) liver. Symbols: measured compound concentrations in influent (hollow) and effluent (filled) perfusate; dashed lines: mean concentrations FIG. 3. Open in new tabDownload slide Typical concentration-time courses of BD and its metabolites EB, B-diol, and DEB in influent and effluent perfusate of a BD-perfused mouse (A) and rat (B) liver. Symbols: measured compound concentrations in influent (hollow) and effluent (filled) perfusate; dashed lines: mean concentrations In mouse and rat livers with similar BD concentrations in the effluent perfusates (BD concentrations ranged in mouse livers up to 161 μmol/l and in rat livers up to 152 μmol/l), the ratio mouse-to-rat of the EB concentrations in effluent perfusates (mean ± SD, n = 5) was 4.5 ± 2.7, that of the B-diol concentrations was 1.7 ± 0.6, demonstrating that both the EB and B-diol concentrations were higher in mouse than in rat livers. In vivo, at equal BD exposure concentrations, concentrations of EB in blood were between 2 and 8.6 times higher in mice as compared to rats; B-diol concentrations were similar in both species (Filser et al., 2007). DEB was determined in two BD-perfused mouse livers with BD concentrations in the effluent perfusate of 8.2 μmol/l (Fig. 3A) and of 1.1 μmol/l. The average concentrations of the three metabolites in the effluents were 6.2 (EB), 5.1 (B-diol), and 0.49 μmol/l (DEB) at the higher (Fig. 3A) and 2.3 (EB), 3.1 (B-diol), and 0.55 μmol/l (DEB) at the lower BD concentration. The ratio EB/DEB was 12 (higher) and 4.2 (lower BD concentration). In vivo, the EB/DEB ratio was smaller: between 1.5 at 67 ppm BD and 5 at 1270 ppm BD (calculated from the data shown for mice in Figure 5 of Filser et al., 2007). The difference might in part result from the continuous perfusion in vivo, which leads to an additional DEB formation from metabolized EB. Two BD exposures of mouse livers were conducted to investigate whether EBD was a further first-pass metabolite of BD. EBD was found as a minor metabolite representing only about 1% of the metabolic BD elimination (data not shown). In the BD-perfused rat livers, DEB was always below the detection limit of the GC/MSD method used for its analysis. No effort was made to measure metabolically produced EBD. The levels of EB in the influent perfusates of both species result most probably from oxidation of BD by the erythrocytes before entering the liver. Erythrocyte-mediated epoxidation of some olefins had already been reported by several authors (e.g., Belvedere and Tursi, 1981; Catalano and Ortiz de Montellano, 1987). Rates of the metabolic elimination of BD and of the net formation of the metabolites appearing in the effluent perfusates were calculated from the means of the data measured at steady state during the BD perfusions. Figure 4 depicts the rates of BD metabolism from 0 up to about 150 μmol/l BD in the effluent perfusate at steady-state exposure, calculated for a liver of a 25-g mouse (Fig. 4A) or a liver of a 250-g rat (Fig. 4B). Each symbol represents the mean rate of BD metabolism obtained from one liver perfusion experiment. The solid lines were fitted to the data using the function that describes saturation kinetics according to Michaelis and Menten. The obtained Vmax values (μmol/[h•kg]) were 300 (mice) and 90.5 (rats) per kilogram of body weight. The apparent Michaelis constants Kmap (μmol/l perfusate) were 16.3 (mice) and 25.8 (rats). FIG. 4. Open in new tabDownload slide Rate of metabolism of BD in dependence of the BD concentration in the effluent perfusate of once-through BD-perfused livers of mice (A) and rats (B). Symbols: “measured” rates of BD metabolism obtained from multiplying the differences of the means of BD concentrations (e.g., in Fig. 3) with the actual perfusion flow; lines: saturation kinetics according to Michaelis and Menten fitted to the symbols. FIG. 4. Open in new tabDownload slide Rate of metabolism of BD in dependence of the BD concentration in the effluent perfusate of once-through BD-perfused livers of mice (A) and rats (B). Symbols: “measured” rates of BD metabolism obtained from multiplying the differences of the means of BD concentrations (e.g., in Fig. 3) with the actual perfusion flow; lines: saturation kinetics according to Michaelis and Menten fitted to the symbols. Figure 5 shows for mice (A) and rats (B) the relative net formation rates of EB and B-diol (net formation rate in percent of the actual rate of BD metabolism) versus the corresponding concentrations of BD in the effluent perfusate. The symbols were calculated from the mean values of individual experiments; the lines represent means of the data (solid, EB; dashed, B-diol). In both species, the relative net formation rates of EB and B-diol remained rather constant over the whole BD concentration range, which means that in both species the net formations of both metabolites are subject to the saturation kinetics of BD. (Considering the well-known conjugation of EB with GSH [Csanády et al., 1992; Kreuzer et al., 1991; Sharer et al., 1992], it follows that there is no relevant GSH depletion in the once-through perfused liver.) The percentages of the relative net formations (means ± SD), related to the actual rates of BD metabolism, were for EB 34 ± 8% (n = 6) and for B-diol 42 ± 7% (n = 6) in mouse livers. In rat livers, the corresponding net formations were 17 ± 11% (n = 8) and 44 ± 14% (n = 8). In other words, relatively less EB was formed in the rat liver when compared to the mouse liver; the relative formation of B-diol was about the same in the livers of both species. The combined fractions of EB and B-diol leaving the liver were 76% (mouse) and 61% (rat) of BD metabolized. FIG. 5. Open in new tabDownload slide Net formations of EB and B-diol in percent of the actual rates of metabolism of BD versus the corresponding BD concentration in the effluent perfusate of BD-perfused mouse (A) and rat (B) livers. Symbols: measured, EB (filled circles) and B-diol (hollow triangles); lines: means fitted to the symbols, straight lines: EB, and dashed lines: B-diol. FIG. 5. Open in new tabDownload slide Net formations of EB and B-diol in percent of the actual rates of metabolism of BD versus the corresponding BD concentration in the effluent perfusate of BD-perfused mouse (A) and rat (B) livers. Symbols: measured, EB (filled circles) and B-diol (hollow triangles); lines: means fitted to the symbols, straight lines: EB, and dashed lines: B-diol. Net formation rates of DEB at the BD concentrations of 1.1 and 8.2 μmol/l in the effluent perfusate of mouse livers represented 8.4 and 3.7%, respectively, of the actual rate of BD metabolism. EBD (determined at BD concentrations of 18 and 161 μmol BD/l effluent perfusate) was a minor direct metabolite with net formation rates of 1.6% (low EB concentration) and 0.5% (high EB concentration) of the actual rates of BD metabolism in mouse livers. In analogy to the BD experiments exemplified in Figure 3, a series of once-through perfusion experiments was carried out in mouse and rat livers with the BD daughter metabolites EB, DEB, B-diol, and EBD (only rat livers). In the influent and effluent perfusates, constant concentrations of each of these metabolites were established. The concentrations agreed with those determined in blood of BD-exposed mice and rats (Filser et al., 2007). The results of the present experiments are summarized in Tables 1 and 2. Table 1 shows the ClH and ClIn of EB, DEB, B-diol, and EBD in mouse and rat livers perfused with the corresponding single compound together with the investigated concentration ranges in the effluent perfusate. Mean ClH values of EB and DEB were 81 and 61% of the mean perfusion flow (Q = 0.246 l/h) in a mouse liver and 85% and 73% of that (Q = 1.10 l/h) in a rat liver, indicating that the metabolic elimination of both compounds was flow limited in both species. This becomes also evident from the ClIn values of EB and DEB, which were between 2.5 (DEB in mouse liver) and 13 times (EB in rat liver) larger than the corresponding perfusion flow. The mean ClH values of B-diol and EBD were distinctly smaller than the mean perfusion flows. For B-diol, they represented 53% (mouse liver) and 34% (rat liver) and for EBD 22% (rat liver) of the corresponding mean perfusion flow. Corresponding mean ClIn values were either slightly higher (mouse liver) or distinctly lower than the perfusion flow (rat liver). ClH and ClIn values shown in Table 1 represent mean values, calculated independently of each other. TABLE 1 Metabolic Clearances (Cl) of EB, DEB, EBD, and B-diol Related to Each Compound Concentration in the Perfusate Entering (ClH) or Leaving (ClIn) the Liver of a Mouse (25 g) or a Rat (250 g) Mouse liver Rat liver ClH (l/h) ClIn (l/h) ClH (l/h) ClIn (l/h) Substance Mean ±SDa Mean ±SDa nb cef (μmol/l) Mean ±SDa Mean ±SDa nb cef (μmol/l) EB 0.20 0.03 1.62 0.97 6 4.1 0.93 (0.76, 1.09) 14.0 (12.6, 15.4) 2 0.4 DEB 0.15 (0.11, 0.19) 0.61 (0.22, 1.0) 2 0.55 0.80 (0.75, 0.85) 5.5 (2.6, 8.4) 2 0.15 B-diol 0.13 (0.13, 0.13) 0.28 (0.25, 0.3) 2 10 0.37 — 0.57 — 1 16 EBD ND ND ND ND ND ND 0.24 (0.20, 0.27) 0.34 (0.28, 0.41) 2 6.3 Mouse liver Rat liver ClH (l/h) ClIn (l/h) ClH (l/h) ClIn (l/h) Substance Mean ±SDa Mean ±SDa nb cef (μmol/l) Mean ±SDa Mean ±SDa nb cef (μmol/l) EB 0.20 0.03 1.62 0.97 6 4.1 0.93 (0.76, 1.09) 14.0 (12.6, 15.4) 2 0.4 DEB 0.15 (0.11, 0.19) 0.61 (0.22, 1.0) 2 0.55 0.80 (0.75, 0.85) 5.5 (2.6, 8.4) 2 0.15 B-diol 0.13 (0.13, 0.13) 0.28 (0.25, 0.3) 2 10 0.37 — 0.57 — 1 16 EBD ND ND ND ND ND ND 0.24 (0.20, 0.27) 0.34 (0.28, 0.41) 2 6.3 Note. ClH, hepatic clearance; ClIn, intrinsic clearance; cef, concentration in effluent perfusate up to which clearance was determined; ND, no data. a When n = 2, single values are given in brackets instead of SD, when n = 1, a dash is plotted. b Number of perfusion experiments. Open in new tab TABLE 1 Metabolic Clearances (Cl) of EB, DEB, EBD, and B-diol Related to Each Compound Concentration in the Perfusate Entering (ClH) or Leaving (ClIn) the Liver of a Mouse (25 g) or a Rat (250 g) Mouse liver Rat liver ClH (l/h) ClIn (l/h) ClH (l/h) ClIn (l/h) Substance Mean ±SDa Mean ±SDa nb cef (μmol/l) Mean ±SDa Mean ±SDa nb cef (μmol/l) EB 0.20 0.03 1.62 0.97 6 4.1 0.93 (0.76, 1.09) 14.0 (12.6, 15.4) 2 0.4 DEB 0.15 (0.11, 0.19) 0.61 (0.22, 1.0) 2 0.55 0.80 (0.75, 0.85) 5.5 (2.6, 8.4) 2 0.15 B-diol 0.13 (0.13, 0.13) 0.28 (0.25, 0.3) 2 10 0.37 — 0.57 — 1 16 EBD ND ND ND ND ND ND 0.24 (0.20, 0.27) 0.34 (0.28, 0.41) 2 6.3 Mouse liver Rat liver ClH (l/h) ClIn (l/h) ClH (l/h) ClIn (l/h) Substance Mean ±SDa Mean ±SDa nb cef (μmol/l) Mean ±SDa Mean ±SDa nb cef (μmol/l) EB 0.20 0.03 1.62 0.97 6 4.1 0.93 (0.76, 1.09) 14.0 (12.6, 15.4) 2 0.4 DEB 0.15 (0.11, 0.19) 0.61 (0.22, 1.0) 2 0.55 0.80 (0.75, 0.85) 5.5 (2.6, 8.4) 2 0.15 B-diol 0.13 (0.13, 0.13) 0.28 (0.25, 0.3) 2 10 0.37 — 0.57 — 1 16 EBD ND ND ND ND ND ND 0.24 (0.20, 0.27) 0.34 (0.28, 0.41) 2 6.3 Note. ClH, hepatic clearance; ClIn, intrinsic clearance; cef, concentration in effluent perfusate up to which clearance was determined; ND, no data. a When n = 2, single values are given in brackets instead of SD, when n = 1, a dash is plotted. b Number of perfusion experiments. Open in new tab TABLE 2 Daughter Metabolites in the Effluent Perfusates in Once-through Perfusions of Mouse or Rat Livers with the Parents EB, DEB, or B-diol (Net Formation Given in Percent of the Metabolic Elimination Rate of the Corresponding Parent) Mouse liver—net formation (%)a Rat liver—net formation (%)b Substance Mean ±SDc nd Mean ±SDc nd Daughters of EB     B-diol 52 24 4 79 5.9 3     DEB 2.1 0.9 5 0.54 (0.52, 0.56) 2     EBD 6 — 1 3.2 (4.7, 1.7) 2 Daughter of DEB     EBD 30 (15, 45) 2 56 (69, 44) 2 Daughter of B-diol     EBD ND ND ND 4 — 1 Mouse liver—net formation (%)a Rat liver—net formation (%)b Substance Mean ±SDc nd Mean ±SDc nd Daughters of EB     B-diol 52 24 4 79 5.9 3     DEB 2.1 0.9 5 0.54 (0.52, 0.56) 2     EBD 6 — 1 3.2 (4.7, 1.7) 2 Daughter of DEB     EBD 30 (15, 45) 2 56 (69, 44) 2 Daughter of B-diol     EBD ND ND ND 4 — 1 Note. ND, no data. a Determined at and below parent concentrations in the effluent perfusate of 4.1 μmol/l (EB) and 0.55 μmol/l (DEB). b Determined at and below parent concentrations in the effluent perfusate of 0.57 μmol/l (EB), 0.15 μmol/l (DEB), or at a concentration of the parent B-diol in the effluent perfusate of 16 μmol/l. c When n = 2, single values are given in parenthesis instead of SD and when n = 1, a dash is plotted. d Number of perfusion experiments. Open in new tab TABLE 2 Daughter Metabolites in the Effluent Perfusates in Once-through Perfusions of Mouse or Rat Livers with the Parents EB, DEB, or B-diol (Net Formation Given in Percent of the Metabolic Elimination Rate of the Corresponding Parent) Mouse liver—net formation (%)a Rat liver—net formation (%)b Substance Mean ±SDc nd Mean ±SDc nd Daughters of EB     B-diol 52 24 4 79 5.9 3     DEB 2.1 0.9 5 0.54 (0.52, 0.56) 2     EBD 6 — 1 3.2 (4.7, 1.7) 2 Daughter of DEB     EBD 30 (15, 45) 2 56 (69, 44) 2 Daughter of B-diol     EBD ND ND ND 4 — 1 Mouse liver—net formation (%)a Rat liver—net formation (%)b Substance Mean ±SDc nd Mean ±SDc nd Daughters of EB     B-diol 52 24 4 79 5.9 3     DEB 2.1 0.9 5 0.54 (0.52, 0.56) 2     EBD 6 — 1 3.2 (4.7, 1.7) 2 Daughter of DEB     EBD 30 (15, 45) 2 56 (69, 44) 2 Daughter of B-diol     EBD ND ND ND 4 — 1 Note. ND, no data. a Determined at and below parent concentrations in the effluent perfusate of 4.1 μmol/l (EB) and 0.55 μmol/l (DEB). b Determined at and below parent concentrations in the effluent perfusate of 0.57 μmol/l (EB), 0.15 μmol/l (DEB), or at a concentration of the parent B-diol in the effluent perfusate of 16 μmol/l. c When n = 2, single values are given in parenthesis instead of SD and when n = 1, a dash is plotted. d Number of perfusion experiments. Open in new tab Table 2 presents the net metabolic formation rates of the metabolites derived from EB, DEB, or B-diol in percent of the elimination rates of the parent compounds (EB, DEB, or B-diol) in perfused rodent livers. In both species, by far, the predominant metabolite of EB was the hydrolysis product B-diol, which represented a higher percentage in rat than in mouse livers. DEB and EBD were formed in the EB-perfused livers too, DEB in rat livers representing only one-fourth of the percentage determined in mouse livers. The relative net formation of EBD from EB in mouse liver effluents was about twice as high as in rat liver effluents, which hints to the relevance of DEB as an EBD precursor in the EB-perfused liver. This conclusion becomes even more evident when comparing the DEB and B-diol perfusions in which only metabolically produced EBD was detected. In the rat liver, EBD represented only a minor B-diol metabolite showing that other pathways are quantitatively much more relevant for the metabolic elimination of B-diol than CYP-mediated epoxidation. DISCUSSION In comparison with in vivo or in vitro methods, the perfused liver system is laborious since toxicokinetic data gained in one liver are valid for a single exposure concentration only. Consequently, the study of concentration-dependent processes required considerable experimental effort. However, the present method gave most relevant new information based on a series of quantitative data on the metabolic fate of the highly important industrial chemical BD. The advantage of the perfused liver system compared to current in vitro methods is the near-to-in vivo situation. Perfusion limitations of metabolism will occur as in the whole animal, and different metabolic pathways can be investigated in parallel, for instance the intrahepatic first-pass metabolism. No artificial separation of metabolic steps and no addition of coenzymes resulting in more or less nonphysiological conditions are required. Considering this, the perfused liver system will be superior to microsomal systems when studying formation (and also elimination) of stereoisomeric BD metabolites (Nieusma et al., 1997) that may have quantitative and qualitative different toxicological effects. The bifunctional BD metabolite DEB forms intra- and interstrand DNA-DNA cross-links the degrees of which depended on the stereoisomer applied (Park et al., 2005; Millard et al., 2006). Enantiospecific reactions were shown with chiral epoxides as small as propylene oxide (Peter et al., 1991). BD metabolism shows saturation kinetics as had been demonstrated in microsomal incubations (rats, Bolt et al., 1983; mice, rats, and humans, Csanády et al., 1992, Filser et al., 1992) and in vivo (rats, Bolt et al. 1984; rats and mice, Kreiling et al., 1986; Medinsky et al., 1994). For in vivo conditions, Vmax values were either estimated by extrapolating in vitro data or by fitting toxicokinetic models to concentration-time courses measured in mice and rats that were exposed to BD in closed chambers. The values used in physiological toxicokinetic models to describe Vmax of BD in livers of mice were between 256 and 400 μmol/(h•kg) (Csanády et al., 1996; Evelo et al., 1993; Johanson and Filser, 1993, 1996; Kohn, 1997; Kohn and Melnick, 2001; Medinsky et al., 1994; Sweeney et al., 1997). In rats, the corresponding values were between 62 and 220 μmol/(h•kg) (Csanády et al., 1996; Evelo et al., 1993; Filser et al., 1993; Johanson and Filser, 1993, 1996; Medinsky et al., 1994; Kohn and Melnick, 2001; Sweeney et al., 1997). The in vivoKmap values used by the same authors were derived from values measured in microsomal incubations or from model optimization using BD exposure data in closed chambers. They were between 2.0 and 26 and between 3.75 and 36 μmol/l blood in livers of mice and rats, respectively. The Vmax and Kmap values obtained in the present work from measurements directly in livers were within the ranges of those hitherto used. Qualitatively, the species difference of the parameter values agrees with that used in the published physiological toxicokinetic models. The ClIn of BD, obtained as the ratio of Vmax to Kmap, was 7.5/16.3 = 0.46 l/h in the liver of a 25-g mouse and 22.6/25.8 = 0.88 l/h in the liver of a 250-g rat. The ClH values, calculated from ClIn and the perfusion flow Q using the equation ClH = (Q•ClIn)/(Q + ClIn), given in Wilkinson and Shand (1975), were 0.16 l/h in the mouse and 0.49 l/h in the rat liver. A comparison of the ClH values with the species-specific mean Q values shows that in vivo during each liver perfusion, at least 35 % (mouse) and 55% (rat) of BD entering this organ should leave it unchanged, remaining systemically available. Filser and Bolt (1984) measured the exhalation of metabolically formed EB into the chamber atmosphere in rats which were exposed in closed chambers to BD concentrations > 2000 ppm. The authors observed that the systemic availability of EB was only 20% of that expected when assuming the first step of BD biotransformation would result in EB, exclusively. They suggested an intrahepatic first-pass effect as causing the reduced bioavailability. When physiologically modeling EB blood concentrations in BD-exposed rodents, this interpretation was taken into account. A part of EB formed from BD within the endoplasmic reticulum of the hepatocyte was modeled to be hydrolyzed in situ, in agreement with an assumption of the existence of an association of the CYP system with microsomal EH (Oesch and Daly, 1972), later extended experimentally by Etter et al. (1991). The residual EB entered the cytosol of the cell where a further part was conjugated with GSH before the remaining EB left the liver and became systemically available (Csanády et al., 1996; Johanson and Filser, 1993, 1996). A later developed more complex model (Kohn and Melnick, 2001) was based on the same considerations. In the model of Sweeney et al. (1997), which was based on an earlier version (Medinsky et al., 1994), it was assumed that oxidative BD metabolism resulted in other metabolites in addition to EB. Only a fraction of the metabolic BD elimination of 0.192 (mouse) and of 0.241 (rat) was considered to represent the formation of EB. Both exposure-independent fractions were obtained by fitting the physiological model to the EB blood data measured by Himmelstein et al. (1994) in mice and rats exposed to constant BD concentrations of 62.5, 625, and 1250 ppm. The present data show the fractions of metabolized BD that leave the livers of mice and rats as EB (Fig. 5). More EB was detected in the effluent perfusate of the mouse liver in agreement with its higher CYP activity to BD, if compared to the rat liver, as evidenced by higher per gram liver weight–normalized Vmax and smaller Km values (this work and measurements in hepatic microsomal incubations: Csanády et al., 1992; Filser et al., 1992). In both species, there is a considerable first-pass metabolism leading predominantly to B-diol and in the mouse liver even to DEB and EBD. However, considering the sums of the investigated metabolites, substantial gaps of about 15–20% (mice) and 40% (rats) in the first-pass metabolism of BD are recognizable. They may contribute to the GSH conjugates with daughter products of B-diol and with EB; derivatives of such adducts had been found in urine of BD-exposed rats, mice, hamsters, monkeys, and humans (e.g., Bechtold et al., 1994; Nauhaus et al., 1996; Richardson et al., 1999; Sabourin et al., 1992). The oxidation of B-diol to hydroxymethylvinyl ketone (Krause et al., 2001) was suggested to be a prominent metabolic fate of B-diol in both rodent species, based on measurements of mercapturic acid urinary metabolites of B-diol (Sprague and Elfarra, 2004). To a small portion, B-diol is also epoxidized to EBD as was detected in a B-diol–perfused rat liver (Table 2). In addition, further catabolism of BD metabolites (e.g., to CO2, Richardson et al., 1999) may contribute to the gaps in the first-pass metabolism of BD. The present findings demonstrating that in BD-perfused livers the pathways resulting from EB conjugation with GSH represent a smaller portion than those following EB hydrolysis are not in conflict with in vivo measurements. Richardson et al. (1999) found that of the inhaled BD doses, 16% (mice) and 8% (rats) in urine was derived from direct GSH conjugation of EB in animals 42 h after nose-only inhalation exposure to 200 ppm [2,3-14C]BD for 6 h. Of the inhaled doses, 26% (mice) and 58% (rats) can be related to EB hydrolysis when summing up the yields of exhaled CO2, of radioactivity still detected after the 42-h time span in the carcass, and of the urinary metabolites from EB hydrolysis. The residual percentages could not be ascribed to one of both pathways. In EB perfusions, the predominant metabolite was B-diol in livers of both species. For the conjugation with GSH, a maximum of 42% (mouse) and of 17% (rat) was left. This finding does not disagree with the results of an in vivo study conducted by Richardson et al. (1998). From ip administered radioactively labeled EB doses (1–50 mg/kg), 52% were detected in urine of mice and 50% in urine of rats (mean values). In addition, 3% (mice) and 5% (rats) were found in feces, 48 h after the injection. Of the urinary radioactivity, 7% (mice) and 20% (rats) were related to the hydrolysis of EB and 68% (mice) and 58% (rats) to direct GSH conjugation of EB. In other words, only 35 and 29% of the administered EB doses were detected in urine of mice and rats, respectively, as derivatives of the conjugation of EB with GSH. Kemper et al. (2001) investigated the metabolism of EB by freshly isolated mouse or rat hepatocytes at 37°C during 45 min of exposures to initial EB concentrations in the hepatocyte suspension of between 5 and 250 μmol/l. From the concentration-time courses of the metabolites B-diol, the GSH conjugate of EB (GSBMO), and “total DEB” (the sum of meso-DEB and racemic DEB, i.e., DEB according to the use in the present work), areas under the concentration-time curves (AUCs) were calculated from 0 until 45 min. Not in contradiction to our data and to that of Richardson et al. (1998), the percentage of GSH conjugation was similar to that of hydrolysis in mice (except the lowest dose) and less compared to hydrolysis in rats. AUCs of total DEB were higher in the hepatocyte suspensions of mice than in those of rats. In 14C-EB–treated mice and rats, Richardson et al. (1998) could not detect derivatives of DEB and EBD. However, the present data show clearly that both metabolites are formed in EB-perfused livers of both species. Interestingly, in mice, a higher portion of DEB is derived from BD than from EB, whereas in rats DEB seems to be formed predominantly from EB (to a small percentage) when entering the liver. In DEB-perfused livers of mice and rats, EBD was a major metabolite of this epoxide. In rat livers, the relative net formation of EBD was almost twice as high as in mouse livers being qualitatively in agreement with the almost five times higher EH activity (Vmax/Kmap) toward DEB in rat liver microsomes as compared to mouse liver microsomes (Boogaard and Bond, 1996). However, as calculated from the above presented results, the main pathway from BD to EBD in the mouse liver proceeds directly from BD (1%) and via EB (2%) and probably B-diol (not quantified) summing up to a net formation rate related to the BD metabolism of 3%, at least. Not more than 2.7% can result from free DEB detected as a result from BD metabolism in the effluent of BD mouse liver perfusates. A corresponding calculation for the rat liver yields net EBD formations related to the rates of BD metabolism of 2.8% (from BD via B-diol and EB) and of 0.05% from the resulting DEB in the effluent. Summing up, only about 6 and 3% are expected to result from BD metabolism in mouse and rat livers, respectively. Interestingly, Richardson et al. (1999) ascribed 6.7% (mouse) and 4.1% (rat) of the inhaled BD doses to urinary trihydroxybutyl mercapturic acids as being derived from EBD and DEB. In vivo at BD exposure concentrations below about 700 ppm, EBD is quantitatively the most abundant BD epoxide metabolite in blood of both species (Filser et al., 2007). This finding might, at least in part, result from the slow metabolic elimination of EBD as compared to those of DEB and EB. In the perfused rat liver, ClIn of EBD is 16 and 41 times slower than that of DEB and EB, respectively (Table 1). ClHs of EB and DEB can be compared to systemic clearances of both compounds obtained in blood of male Sprague-Dawley rats (Valentine et al., 1997). Following iv administrations of EB doses of 71, 143, and 286 μmol/kg body weight and of a DEB dose of 523 μmol/kg body weight, the authors determined nonsignificantly different clearances of 104, 114, and 67 ml/(min•kg) for EB and of 76 ml/(min•kg) DEB. For a 250-g rat, these clearances yield values of between 1.0 and 1.7 l/h for EB and of 1.14 l/h for DEB, means being 54 and 43% higher than those in the perfused livers (present work). In agreement with Valentine et al. (1997), we conclude that extrahepatic elimination might be causing the increased systemic clearances if compared to the blood flow through the livers of both species. In addition to the ClHs of EB and DEB in rats, ClH of B-diol, gained in the perfused mouse liver, can be compared with in vivo data published by Kemper et al. (1998). These authors measured the disappearance of B-diol in plasma of male B6C3F1 mice following ip injections of diverse doses, the lowest of which being 10 mg/kg body weight (=114 μmol/kg, far above the highest B-diol concentration of 24 μmol/l in the influent perfusate in the present study). When plotting semi-logarithmically plasma concentrations versus time, Kemper et al. (1998) found dose-dependent slopes. The lowest dose resulted in the highest slope yielding a plasma clearance of 5658 ± 695 ml/(h•kg) corresponding to a clearance of 0.141 ± 0.017 l/h in a mouse of 25 g. In the present work, ClH of B-diol in the perfused mouse liver showed almost the same value. This would mean B-diol metabolism to occur in the liver almost quantitatively when assuming the distribution of B-diol between plasma (the matrix used in Kemper et al., 1998) and blood (or the erythrocytes containing perfusate) to be close to unity. In summary, the studies in perfused mouse and rat livers clarified quantitative aspects on the species-specific role of the liver with respect to the metabolism of BD and the formation as well as the metabolism of toxicologically relevant metabolites. In addition, there is evidence for qualitative differences in the formation of these metabolites between mouse and rat livers. There was explicit DEB formation in the BD-perfused mouse liver whereas DEB was not detectable in the BD-perfused rat liver. This finding implies not only species differences in the intracellular first-pass metabolism of EB when produced from BD by in situ hydrolysis but also points to an in situ epoxidation of EB to DEB in the mouse liver which may be much less or not existent in the rat liver. The species-specific extent of both effects might depend on species differences in the active site of CYP2E as was assumed by Lewis et al. (1997). This difference might also be relevant concerning the metabolism of other olefins. Therefore, the present work might lead to a better understanding of differences in the hepatic metabolism of olefins between mice and rats. The data and the parameters presented will be the most relevant for the development of a physiological toxicokinetic model that will serve as a more reliable basis for risk estimation of BD than the models published so far. FUNDING Olefins Panel of the American Chemistry Council. The authors thank Dr Judith Baldwin for the quality assurance reviews. 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TI - Quantitative Investigation on the Metabolism of 1,3-Butadiene and of Its Oxidized Metabolites in Once-through Perfused Livers of Mice and Rats
JF - Toxicological Sciences
DO - 10.1093/toxsci/kfp297
DA - 2010-03-01
UR - https://www.deepdyve.com/lp/oxford-university-press/quantitative-investigation-on-the-metabolism-of-1-3-butadiene-and-of-0Tw1vPgfIJ
SP - 25
EP - 37
VL - 114
IS - 1
DP - DeepDyve
ER -