Kinetic assessment of persistent halogenated xenobiotics in cell culture models: comparison of mono- and poly-halogenated compounds.

We evaluated the suitability of single and multiple cell type cultures as model systems to characterise cellular kinetics of highly lipophilic compounds with potential ecotoxicological impact. Confluent mono-layers of human skin fibroblasts, rat astrocytoma C6 cells, non-differentiated and differentiated mouse 3T3 cells were kept in culture medium supplemented with 10% foetal calf serum. For competitive uptake experiments up to four different cell types, grown on glass sectors, were exposed for 3h to (14)C-labelled model compounds, dissolved either in organic solvents or incorporated into unilamellar lecithin liposomes. Bromo-, or chloro-benzenes, decabromodiphenylether (DBP), and dichlorodiphenyl ethylene (DDE) were tested in rather high concentration of 20 microM. Cellular toxicity was low. Compound levels were related to protein, DNA, and triglyceride contents. Cellular uptake was fast and dependent on physico-chemical properties of the compounds (lipophilicity, molecular size), formulation, and cell type. Mono-halogenated benzenes showed low and similar uptake levels (=low accumulation compounds). DBP and DDE showed much higher cellular accumulations (=high accumulation compounds) except for DBP in 3T3 cells. Uptake from liposomal formulations was mostly higher than if compounds were dissolved in organic solvents. The extent of uptake correlated with the cellular content of triglycerides, except for DBP. Uptake competition between different cell types was studied in a sectorial multi-cell culture model. For low accumulation compounds negligible differences were found among C6 cells and fibroblasts. Uptake of DDE was slightly and that of DBP highly increased in fibroblasts. Well-defined cell culture systems, especially the sectorial model, are appropriate to screen for bioaccumulation and cytotoxicity of (unknown) chemical entities in vitro.

N-Acetyl-procainamide kinetics in the elderly.

Plasma and saliva N-acetyl-procainamide (NAPA) concentrations were measured by high-power liquid chromatography (HPLC) after intravenous infusion of 750 mg to 14 elderly patients (x age = 69 yr). The plasma NAPA disappearance curve can best be described by a two-compartment body model. Mean total body clearance was 10.6 1/hr, Vdss 125.8 1, and terminal half-life (t 1/2) 8.8 hr. A nonrenal clearance of 2.72 1/hr was calculated, that is, 19% of the expected total body clearance with normal kidney function. Saliva concentrations show huge inter- and intraindividual variability and are probably not usable for NAPA monitoring in older patients.

Defective bile acid transport in an animal model of defective debrisoquine hydroxylation.

Bile acid transport in female DA (dark Aguti) rats, a model for debrisoquine hydroxylation deficiency in man, was investigated. Compared to hydroxylation competent male DA and Sprague-Dawley rats of either sex, the female DA rat had a significantly lower taurocholate maximal secretory rate in vivo. Studies in the perfused liver showed this to be due to a decreased extraction efficiency during exogenous taurocholate loading. To characterize further the defect, taurocholate uptake velocity into isolated hepatocytes was studied. This showed a decreased maximal uptake velocity in the female DA rat (P less than 0.02). Whether this defect in bile acid uptake is related to the defective debrisoquine hydroxylation, remains to be established.

In vivo hydroxylation of 3H-acetanilide--evaluation of a new radiospirometric method in the rat.

The proposed in vivo methodology for the investigation of hydroxylation rates consists of of the i.v. administration of tritiated substrates and the collection of tritiated water (HTO) from exhaled air as a measure of HTO accumulation in body water. Specifically, HTO was assessed in exhaled water after i.v. administration of 3H-acetanilide. Over a wide range the half lives of accumulation of HTO in exhaled water (T50) were almost identical with the half lives of elimination of 3H-acetanilide in blood, evaluated by an inverse isotope dilution method (r = 0.96, N = 18). Average T50 amounted to 29 min in controls, was reduced to 20 min after enzyme induction by phenobarbital or 3-methylcholanthrene, and prolonged to 45, 46 and 66 min after bile duct ligation, portacaval shunt and a single dose of ethanol, respectively. It is concluded that the chosen pharmacokinetic approach corrects for the NIH-shift and the results adequately reflect changes in acetanilide hydroxylation related to enzyme induction or inhibition and to liver pathology.

Determination of bile acids in serum by capillary gas-liquid chromatography.

A glass capillary column and an appropriate relatively simple procedure for sample preparation have been developed for determination of serum bile acids. Sample preparation involved extraction with Amberlite XAD-2, solvolysis of sulfates, enzymatic hydrolysis with cholylglycine hydrolase, methylation and silylation. Because of complete chromatographic separation of bile acid trimethylsilylether derivatives from cholesterol on the capillary column, an additional step for elimination of cholesterol could be omitted. Trimethylsilylether derivatives were separated on a 20 meter x 0.3 mm i.d. glass capillary column covered with a crystal layer of barium carbonate and coated with polyethyleneglycol 20,000 as liquid phase according to Grob, K. and Grob, G. (1976) J. Chromatogr.125, 471--485, and Grob, K., Grob, G. and Grob, Jr., K., (1977) Chromatographia 10, 181--187. Overall recovery of the major human conjugated bile acids ranged from 86 to 89%. Reproducibility of bile acid determination was satisfactory in both normal and pathological serum with elevated bile acid concentrations (coefficient of variation 7.6 to 10.0%). The mean concentrations of cholic, deoxycholic, chenodeoxycholic and lithocholic acid in the serum of healthy subjects were 0.9, 1.0, 1.7 and 0.2 mumol/l in males, and 1.0, 0.8, 1.4 and 0.2 mumol/l in females.

A highly specific 125I-radioimmunoassay for cholic acid conjugates.

Several modifications of the immunization procedure permitted development of a highly specific radioimmunoassay (RIA) for cholic acid conjugates. Antiserum was produced in guinea pigs using cholic acid-thyroglobulin complex as immunogen. 125-I-Cholyglycylhistamine was prepared as radioactive ligand according to a modification of the method of Spenney et al. (Spenney, J.G., Johnson, B.J., Hirschowitz, B.I., Mihas, A.A. and Gibson, R. (1977) Gastroenterology 72, 305--311). The association constant of the antisera to taurocholic acid was 1.8 x 10(7) l/mol, the working range of the assay between 9.5--890 pmol. Cross-reactivities of the antiserum to bile acids other than cholic acid species were less than 3%, which is lower than for any published bile acid RIA. Concentrations of cholic acid conjugates in sera obtained from 17 healthy fasting volunteers ranged from 0.4--1.9/mumul/l.

Determination of polychlorinated dibenzo-p-dioxins and dibenzo-furans in solid residues from wood combustion by HRGC/HRMS.

PCDD/PCDF were determined in solid samples from wood combustion. The samples included grate ashes, bottom ashes, furnace ashes as well as fly and cyclone ashes. The solid waste samples were classified into bottom and fly ash from native wood and bottom and fly ash from waste wood. For each of the four classes concentration distribution patterns from individual congeners, the sums of PCDD/PCDF and the international toxicity equivalents (I-TEQ) values are given. The I-TEQ levels of fly ash from waste wood burning can be approximately up to two thousand times higher than the values from fly ashes of natural wood. The I-TEQ levels in bottom ashes from waste wood combustion systems are as low as the corresponding ashes from the combustion of native wood. Grate ash samples from waste wood combustion systems with low carbon burnout show high levels of PCDD/PCDF.

The elaboration of the 'Stockholm convention' on persistent organic pollutants (POPs): a negotiation process fraught with obstacles and opportunities.

The conclusion in December 2000 of the negotiations for the 'Stockholm Convention' can clearly be labeled as a success. The Convention text was negotiated in merely five sessions of the Intergovernmental Negotiating Committee (INC) and accomplished after its fifth session despite the fact that numerous controversial issues, such as the inclusion of new substances under the ambit of the Convention, the acknowledgement of the precautionary principle or--clearly most controversial--the financing mechanisms, remained to be resolved. This paper attempts to provide a somewhat impressionistic account of the negotiations leading to the conclusion of the 'Stockholm Convention' as experienced by the members of the Swiss delegation participating in the negotiations of the INC. Besides a brief overview on the 'history' of the negotiations, it will focus on some issues of special interest--and controversy--to the negotiators, and finally attempt to provide an outlook on the future of the work performed by the INC and the implementation of the Convention. Issues of special interest are environmental policy issues, capacity building and financing, trade-related issues, precautionary principles, and technical and scientific issues.

Clearance of atrazine in soil describing xenobiotic behavior.

The primary aim of this study was to evaluate the "clearance concept" as a tool for describing the behavior of xenobiotic movement into and through soils. As an example, degradation of 2-chloro-4-ethylamino-6-isopropylamino-s-triazine (atrazine) with the formation of metabolites 2-chloro-6-isopropylamino-s-triazine (desethylatrazine) and 2-chloro-4-ethylamino-s-triazine (desisopropylatrazine) was investigated. Atrazine was sprayed post-emergently in doses of 0.125 or 0.5 g active ingredient/m(2) each on four test plots. Soil type was a sandy-loam, on which corn (Zea mays L.) was cultivated. Soil samples were taken as cores of 0.2 m depth 0, 1, 2, 4, 8, 12, 16 and 20 weeks after application of atrazine, and analyzed by HPLC. Soil concentrations of atrazine were highly correlated (r=0.993, p< 0.001) between the two applications of 0.125 g/m(2) and 0.5 g/m(2). Up to 50% of the atrazine was measured as metabolites during the whole vegetation period. Clearance of atrazine from soil was calculated as the total load of atrazine divided by the area under the soil atrazine concentration time curve. Soil atrazine clearance was calculated as 5.13 +/- SD 1.10 and 5.17 +/- SD 1.02 liter of soil per day for doses of 0.125 g/m(2) and 0.5 g/m(2), respectively (from a "soil unit" of 1 × 1 × 0.2 meter). The clearance concept might be a tool for risk assessment of xenobiotics.

Analysis of bile acids in serum and bile by capillary gas-liquid chromatography.

Various liquid phases for glass capillary columns have been evaluated for gas-liquid chromatographic analysis of methyl ester trimethylsilylether derivatives of bile acids from serum and bile. Bile acid analysis is rapid and exhibits high separation efficiency with a 20 X 0.3 mm glass capillary column whose internal surface is covered with a crystal layer of barium carbonate and coated with polyethyleneglycol 20000 as liquid phase according to Grob et al.

Bioavailability determination of lidocaine by capillary gas chromatography ammonia chemical ionization mass spectrometry.

A method was developed to measure bioavailability of lidocaine by simultaneous peroral and intravenous dosing. Lidocaine hydrochloride corresponding to 125 mg base was given perorally. Simultaneously, 30 mg of deuterated lidocaine-d3 were injected intravenously. Blood samples were taken at intervals for 270 min. Plasma samples were spiked with mepivacaine hydrochloride as internal standard, alkalinized to pH 11.7 and extracted with diethyl ether. The extracts were analysed by capillary GC ammonia CI MS using a 15 m X 0.32 mm i.d. glass capillary column coated with SE-54. The ion source pressure was 0.4 Torr of ammonia as reagent gas. Quasimolecular ions were monitored at m/z 235, 238 and 247 for lidocaine, lidocaine-d3 and mepivacaine, respectively. Calibration curves were linear from 0.2 to 5.0 nmol lidocaine ml-1 plasma. Interday reproducibility of this method was 6.9% for lidocaine-d3 (n = 16; 1.90 +/- 0.13 nmol ml-1). Bioavailability of lidocaine in 5 normal volunteers ranged from 26 to 36% (mean 31 +/- SD 5%) and in a cirrhotic with an end-to-side portacaval shunt it approached 100%, as anticipated. The method is well suited for determination of bioavailability of lidocaine after simultaneous administration of rather small and safe doses both intravenously and perorally.

Altered bile acid profiles in duodenal bile and urine in diabetic subjects.

The bile acid composition in duodenal bile was analysed in 22 diet-treated and 11 insulin-treated middle-aged patients with diabetes mellitus and in 20 normoglycaemic controls. In 10 subjects with diabetes mellitus the bile acid profile in urine was also investigated. In the non-insulin-dependent diabetic patients the percentage of cholic acid was reduced and that of deoxycholic acid increased. As a highly significant finding there was a three-fold increase of the percentage of 12-ketolithocholic acid in duodenal bile in non-insulin-dependent diabetics, whereas the bile acid composition in insulin-dependent diabetics was similar to that in a control group. The percentage of 12-ketolithocholic acid in duodenal bile was positively correlated to the percentage in urine. In nine of the subjects studied, 12-ketolithocholic acid was the major individual bile acid in urine. It constituted 36.3 +/- 4.4% of the bile acids analysed and the excretion was 6.1 +/- 2.3 mumol 24 h-1. Together with 3 alpha, 12 beta-dihydroxy-5 beta-cholanoic acid it was predominantly present in the glycine conjugate fraction, whereas in bile its conjugation was similar to that of the other bile acids. The results may reflect an increased formation of secondary bile acids from cholic acid combined with a metabolic disturbance in non-insulin-dependent diabetics affecting the oxidoreduction of bile acids at C-12.

Identification of 27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24 xi, 25 xi,26-hexol and partial characterization of the bile alcohol profile in urine.

The nature of the bile alcohols present in urine of an infant with neonatal cholestasis has been investigated. Urine was extracted with Sep-Pak C18 cartridges and a glucuronide fraction was isolated by ion exchange chromatography on Lipidex-DEAP. Following enzymatic hydrolysis and purification on Lipidex-DEAP, the bile alcohols were isolated by high performance liquid chromatography. Fourteen compounds were studied by a combination of microchemical reactions and capillary column gas-liquid chromatography-mass spectrometry. Both C26 and C27 bile alcohols were present. Among the former, three additional isomers of the previously identified 27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24 xi,25 xi-pentol were detected. A new C26 bile alcohol, 27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24 xi,25 xi,26 -hexol, was identified, and a 27-norcholestane-pentolone with hydroxyl groups at C-24 and C-25 and a keto group in the ring system was partially characterized. The C27 bile alcohols consisted of cholestanepentols, -tetrolones, and -pentolones. 5 beta-Cholestane-3 alpha,7 alpha,12 alpha,25,26-pentol (5 beta-bufol), one of its isomers and an isomer of cholestane-3,7,12,24,26-pentol were present. Two cholestanetetrolones and two cholestanepentolones having the keto group in the ring system were partially characterized. The hydroxyl groups in the side chain of the tetrolones were at C-24,26 and C-25,26, respectively, whereas the pentolones had hydroxyl groups at C-24,25 and C-25,26, respectively. The excretion of glucuronidated bile alcohols in urine is suggested to reflect an alternative metabolism of intermediates in the normal biosynthesis of bile acids.

Urinary bile acid and bile alcohol excretion does not reflect the genetic polymorphism of debrisoquine hydroxylation.

Excretion of the major urinary bile alcohol 27-nor-5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 24,25- pentol , and of cholic, chenodeoxycholic, deoxycholic and lithocholic acid was measured in 24 h urine collections of 10 extensive and seven poor metabolizers of debrisoquine. There was no significant difference of the excretion of these cholesterol metabolites between the two groups, indicating that cholesterol hydroxylation to bile alcohols and bile acids is probably not controlled by the same genes responsible for the 'debrisoquine-type' hydroxylation polymorphism.

Cholesterol synthesis in the perfused liver of pregnant hamsters.

Pregnancy is a risk factor for the development of cholesterol gallstones. In pregnant women, biliary cholesterol saturation and secretion are increased. To investigate whether this was due to increased cholesterol synthesis, we studied hepatic cholesterol synthesis in Syrian Golden hamsters. Female controls and animals 10- to 14-days pregnant were studied. The studies were performed in the in situ perfused hamster liver. Cholesterol synthesis was determined by measuring the incorporation of 3H2O added to the perfusate into hepatic, perfusate, and bile cholesterol during a 90-min period. In both pregnant groups, bile flow decreased significantly, but biliary cholesterol concentration increased only in the 14-day pregnant group. The cholesterol synthesis rate averaged (mean +/- SD) 172 +/- 27, 127 +/- 37, and 552 +/- 79 nmol X hr-1 X g liver-1 in controls, 10-day, and 14-day pregnant animals, respectively. The 14-day pregnant animals secreted a markedly higher fraction (47.3 +/- 11.3 vs. 11.1 +/- 13.4%; P less than 0.01) of newly synthesized cholesterol into bile but not into perfusate. Chenodeoxycholate, but not cholate, synthesis rate was decreased in both pregnant groups. We conclude from our studies that hepatic cholesterol synthesis increases towards the end of pregnancy in the hamster and that more newly synthesized cholesterol is secreted into bile at that time. This could at least partially explain the increased biliary cholesterol saturation and secretion observed in women in the third trimenon, and explain pregnancy as a risk factor in the development of cholesterol gallstones.

Monohydroxy bile salt sulfates: tauro-3 beta-hydroxy-5-cholenoate-3-sulfate induces intrahepatic cholestasis in rats.

The biologic effects of sulfation of tauro-3 beta-hydroxy-5-cholenoate and of taurolithocholate were compared. Equimolar amounts (100 nmol/min X 100 g body wt) of the following were administered intravenously to male Sprague-Dawley rats over a 180-min period: taurolithocholate, [14C]taurolithocholate-3-sulfate, tauro-3 beta-hydroxy-5-cholenoate, [14C]tauro-3 beta-hydroxy-5-cholenoate-3-sulfate, its combination with taurocholate, and a saline-albumin solution (control). Sulfation of taurolithocholate and of tauro-3 beta-hydroxy-5-cholenoate only prevented the cholestatic effect of the former. Bile flow during infusion of [14C]tauro-3 beta-hydroxy-5-cholenoate-3-sulfate was reduced by 80% at the end of the experiment. A dose-dependent bile flow reduction was demonstrated. Recovery of the administered bile acid was 3% in urine, 13% in serum, 23% in the liver tissue, and 52% in bile, respectively. Excretion of biliary cholesterol and phospholipids was significantly reduced during the first hour of infusion. Coadministration of taurocholate abolished the cholestatic effect and enhanced the renal excretion of the sulfated bile salt. These data suggest that (a) the cholestatic effect of tauro-3 beta-hydroxy-5-cholenoate-3-sulfate is comparable with or may even exceed the effect of taurolithocholate and (b) although sulfation renders some bile salts more water soluble, it does not prevent the cholestatic effect of all monohydroxy bile salts.

High-performance liquid chromatographic determination of dimethylxanthine metabolites of caffeine in human plasma.

A normal-phase high-performance liquid chromatographic assay of caffeine and its metabolites, theophylline, theobromine and paraxanthine, in human plasma is described. The two internal standards ethyltheophylline and 1,3,7-trimethyluric acid are used simultaneously and cover the range of different polarities from caffeine to the three dimethylxanthines. Plasma (0.5 ml) in the presence of ammonium sulphate is extracted with chloroform--isopropanol (1:1, v/v). The extract is chromatographed with a LiChrosorb Si 60 5-micron column and a mobile phase of dichloromethane containing 2.5% of a formate buffer in methanol. Calibration is performed with six different calibration mixtures which take into account the large plasma concentration differences between caffeine and its metabolites in man. The method is suitable for the simultaneous determination of caffeine and its dimethylxanthine metabolites in plasma of healthy and diseased persons.