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.

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.

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.

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.

Organic micropollutants in Swiss sewage sludge.

Sludges from sixteen municipal and eleven industrial Swiss sewage treatment plants were analysed for adsorbable organic compounds (AOX), chlorinated pesticides, polychlorinated biphenyls and polycyclic aromatic hydrocarbons. Except for the AOX values, which were significantly higher (p < 0.05) in industrial sludges, there was no significant difference between industrial and municipal sludges. The AOX values did not correlate with any of the measured pollutants. The methods employed (capillary GC-MS and GC-electron-capture detection) proved to be suitable for monitoring organic micropollutants in industrial and municipal sewage sludges.

Excessive motor impairment two hours after triazolam in the elderly.

The pharmacokinetics of triazolam 0.25 mg p.o. and psychomotor coordination were compared in nine healthy, elderly volunteers and nine middle aged controls. Motor coordination, as measured by pursuit rotor performance, was impaired in the elderly even before triazolam administration, and in contrast to the controls it deteriorated to a critical level after the drug. Factors associated with the major decrease in psychomotor performance in the elderly volunteers were poor baseline performance, an additional independent-age factor, and the plasma concentration of free triazolam. Although short acting benzodiazepines may have a less detrimental effect on performance on the morning following their intake, there may be serious motor incoordination and falls may occur if the patients have to rise during the night, particularly when the plasma concentration is high, i.e. about 2 h after dosing.

Increased urinary excretion of bile alcohol glucuronides in patients with primary biliary cirrhosis.

The bile alcohol glucuronides in urine of 12 patients with primary biliary cirrhosis (PBC), 10 patients with chronic active hepatitis (CAH), and 6 healthy volunteers were analyzed by capillary gas-liquid chromatography-mass spectrometry. In all subjects studied, the major urinary bile alcohol was found to be 27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,25-pentol (C26 pentol). In PBC patients, the excretion of C26 pentol (main isomer) was significantly increased above values observed in healthy volunteers (mean +/- SD = 5.2 +/- 3.5 mumol/24 h, range 1.0-13.4; versus 0.6 +/- 0.3, range 0.4-1.0). In addition, PBC patients excreted increased amounts of other bile alcohols such as isomers of C26 pentol, pentahydroxylated C27 bile alcohols (5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,25-pentol) and 5 beta-cholestane-3 alpha,7 alpha,12 alpha,25,26-pentol) and a hexahydroxylated C26 bile alcohol (27-nor-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24,25,26-hexol). In CAH patients, the excretion of the C26 pentol main isomer ranged from 0.3 to 2.0 mumol/24 h (mean +/- SD = 0.7 +/- 0.5) and did not significantly differ from that in healthy volunteers. Moreover, the bile alcohol profile was comparable to those found in healthy volunteers and PBC patients. These findings show that total urinary bile alcohol glucuronide excretion is significantly increased in primary biliary cirrhosis. A PBC-specific urinary bile alcohol profile, however, does not exist.

Preserved cytosolic and synthetic liver function in jaundice of severe extrahepatic infection.

We investigated prospectively 9 adult patients with the syndrome of jaundice complicating severe extrahepatic infection both clinically and by quantitative liver function tests. Five patients having severe extrahepatic infection without jaundice were used for comparison. Intraperitoneal infection was found to be a major risk factor for development of jaundice. Jaundice was mainly associated with gram-negative infection, but did not influence survival. Duration of jaundice was dependent on control of the underlying infection. Liver function tests showed a severely deranged organic anion transport, whereas synthetic, cytosolic, and microsomal functions remained preserved. Our study shows that (a) the syndrome of jaundice associated with extrahepatic infection is a functional disorder that is reversible upon control of infection, and that (b) cytosolic, synthetic, and microsomal function is preserved. This may have consequences for both assessing prognosis and clinical management.

A bile alcohol sulfate as a major component in the bile of the small skate (Raja erinacea).

The nature of bile alcohols and bile acids in gall-bladder and hepatic bile from perfused livers of the small skate (Raja erinacea) has been investigated. The main bile alcohol sulfate was isolated by thin-layer chromatography and analyzed by fast atom bombardment mass spectrometry and 13C NMR. Following solvolysis and purification on Lipidex-DEAP, the bile alcohol profile was measured by capillary gas-liquid chromatography-electron impact mass spectrometry. Based on these studies and on comparison with authentic scymmnol sulfate and scymnol, the main bile alcohol was identified as 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24 xi,26,27-hexol sulfate. The mean +/- SD concentration in gallbladder bile from five different skates was 24.6 +/- 8.7 mmol/l. Only 0.1 mmol/l of cholic acid and its conjugates was found in a pool of skate bile. The main bile alcohol sulfate in the bile of the small skate seems to be a metabolic end product, present in a concentration comparable to that of bile salts in mammals.

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.

Acetylation of acetylhydrazine, the toxic metabolite of isoniazid, in humans. Inhibition by concomitant administration of isoniazid.

The effect of isoniazid and its metabolites on the disposition of acetylhydrazine, a toxic metabolite formed from isoniazid, was studied in humans. Acetylhydrazine was administered i.v. with and without prior ingestion of 300 mg of isoniazid. In the studies with isoniazid, 15N2-acetylhydrazine was administered in order to distinguish exogenous acetylhydrazine from the unlabeled acetylhydrazine formed from isoniazid. In each subject (two fast and three slow acetylators) the rate of elimination of acetylhydrazine was similar to the rate of elimination of isoniazid suggesting that the two compounds are subject to the same acetylation polymorphism. In the presence of isoniazid the rate of elimination of acetylhydrazine was consistently lower than in the absence of isoniazid, and the urinary excretion of diacetylhydrazine and the ratio of diacetyl/acetylhydrazine in urine decreased. The data indicate that therapeutic concentrations of isoniazid and its metabolites inhibit the acetylation of acetylhydrazine in humans. The inhibition of this detoxification pathway could contribute to the hepatotoxicity of isoniazid.

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.

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.

Mechanism of the excessive sedative response of cirrhotics to benzodiazepines: model experiments with triazolam.

Mechanisms responsible for disproportional sedation resulting from triazolam administration to patients with cirrhosis were investigated. Ordinary sedative doses (0.25 mg) were given p.o. to 8 cirrhotics and 18 controls. Plasma concentrations of unbound drug were assessed by capillary gas chromatography and equilibrium dialysis. Median apparent oral clearances of unbound triazolam were 14.8 ml per min per kg in cirrhotics and 23.9 ml per min per kg in controls (p less than 0.01). Clearances were significantly correlated with severity of liver disease as assessed by the aminopyrine breath test (Rs = 0.77, n = 17, p less than 0.001). At a time when plasma concentrations of unbound triazolam were the same in both groups, i.e., 2.25 hr after dosing, flicker sensitivity at 5 Hz which was used as an index of CNS performance was impaired by a factor of 3.2 in cirrhotics and 1.4 in controls (p less than 0.01 for group difference). Performance was also significantly lower in cirrhotics with the digit symbol substitution test (p less than 0.05). It is concluded that, in patients with cirrhosis, disproportional sedation after benzodiazepine administration may be due not only to impaired drug elimination, but also to hypersensitivity of the brain.

Increased urinary excretion of toxic hydrazino metabolites of isoniazid by slow acetylators. Effect of a slow-release preparation of isoniazid.

To test the hypothesis that slow acetylators, who may have a greater risk of developing isoniazid hepatitis than rapid acetylators, are exposed to more acetylhydrazine and hydrazine, two toxic metabolites of isoniazid, the urinary excretion of hydrazino metabolites of isoniazid was measured following the ingestion of 300 mg isoniazid. Slow acetylators (n = 7) excreted significantly more isoniazid (32.4 vs 9.2% dose), acetylhydrazine (3.1 vs 1.6% dose), and hydrazine (1.0 vs 0.4% dose) in 24 h than rapid acetylators (n = 5), whereas the excretion of acetylisoniazid and diacetylhydrazine was significantly lower. As the acetylation (i.e. detoxification) of acetylhydrazine is inhibited in the presence of high concentrations of isoniazid, a study was also made of the effect of a slow-release preparation that results in lower plasma concentrations of isoniazid on the production of hydrazino metabolites. The ratio of acetylisoniazid to isoniazid in urine was significantly increased in slow acetylators from 0.84 to 1.02 following administration of the slow release preparation, indicating increased acetylation of isoniazid. However, the excretion of diacetylhydrazine relative to the excretion of acetylhydrazine and hydrazine did not change. It is concluded that exposure to toxic metabolites of isoniazid is increased in slow acetylators. Detoxification of the toxic metabolites was not enhanced by a slow-release preparation of isoniazid.

Bile acids and bile alcohols in two patients with Zellweger (cerebro-hepato-renal) syndrome.

The Zellweger cerebro-hepato-renal syndrome (CHRS) is a rare hereditary disease in which there is a generalized deficiency of peroxisomal function. Liver peroxisomes are important for the conversion of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid into cholic acid, and, consequently, 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid and metabolites of this bile acid precursor accumulate in serum and bile of patients with CHRS. Little is known about the urinary excretion of bile acids in this disease. Using gas chromatography-mass spectrometry we have analyzed serum bile acids and urinary excretion of bile acids and bile alcohols in two Swiss male CHRS patients. As expected, serum concentrations and urinary excretions of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid and 3 alpha,7 alpha,12 alpha,24-tetrahydroxy-5 beta-cholestanoic acid were elevated, which is probably an obligatory finding in CHRS. In addition, the urinary excretion of 1,3,7,12-tetrahydroxy-5 beta-cholanoic acid (a very polar unusual bile acid) was increased (99-1556 nmol/24 h). In contrast, the excretion of the major urinary bile alcohol, 27-nor-5 beta-cholestane-3 alpha, 7 alpha,12 alpha,24,25-pentol was found to be normal. 3 alpha, 7 alpha,12 alpha-Trihydroxy-5 beta-C29-dicarboxylic acid, a metabolite of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid previously believed to be obligatory in CHRS, was found only in one of our 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.