A Degrading Potassium Tablet Mimicking Active Gastric Bleeding in a Computer Tomographic Investigation.

A 54-year-old male patient was admitted to the hospital due to symptoms caused by an intramural hematoma of the descending aorta. In a contrast media-enhanced computed tomography scan performed five days after admission to evaluate dynamics of the hematoma, a hyperdense lesion was seen in the stomach. A suspicion of gastric hemorrhage was raised at the first evaluation. Because the patient's clinical condition and hemoglobin levels were stable, gastroscopy to rule out an aorto-gastric fistula or another type of bleeding was not undertaken. In the secondary evaluation of the history and images, it became clear that the hyperdense lesion mimicking bleeding in the stomach must have been caused by a degrading potassium tablet ingested by the patient five hours before the investigation.

Mobile phone conversations, listening to music and quiet (electric) cars: Are traffic sounds important for safe cycling?

Listening to music or talking on the phone while cycling as well as the growing number of quiet (electric) cars on the road can make the use of auditory cues challenging for cyclists. The present study examined to what extent and in which traffic situations traffic sounds are important for safe cycling. Furthermore, the study investigated the potential safety implications of limited auditory information caused by quiet (electric) cars and by cyclists listening to music or talking on the phone. An Internet survey among 2249 cyclists in three age groups (16-18, 30-40 and 65-70year old) was carried out to collect information on the following aspects: 1) the auditory perception of traffic sounds, including the sounds of quiet (electric) cars; 2) the possible compensatory behaviours of cyclists who listen to music or talk on their mobile phones; 3) the possible contribution of listening to music and talking on the phone to cycling crashes and incidents. Age differences with respect to those three aspects were analysed. Results show that listening to music and talking on the phone negatively affects perception of sounds crucial for safe cycling. However, taking into account the influence of confounding variables, no relationship was found between the frequency of listening to music or talking on the phone and the frequency of incidents among teenage cyclists. This may be due to cyclists' compensating for the use of portable devices. Listening to music or talking on the phone whilst cycling may still pose a risk in the absence of compensatory behaviour or in a traffic environment with less extensive and less safe cycling infrastructure than the Dutch setting. With the increasing number of quiet (electric) cars on the road, cyclists in the future may also need to compensate for the limited auditory input of these cars.

Effect of physical effort on mental workload of cyclists in real traffic in relation to age and use of pedelecs.

To improve cycling safety, insight is required into the factors that contribute to road safety of older cyclists. From the wide range of possible factors, this paper addresses the role of physical effort on mental workload of cyclists with the aim to investigate whether physical effort affects mental workload of cyclists in real traffic in a field experiment. Two instrumented bicycles, a conventional bicycle and a pedelec, were used. Mental workload of cyclists in two age groups - 30-45 years and 65 years and over - was measured by means of a secondary cognitive task requiring the detection and reaction to visual stimuli on a cycle route that varied in physical effort and task complexity. We expected physical effort to impair performance on the secondary task in complex traffic sections and not in simple sections, and that this impairment would be greater for older cyclists because of age related reduced muscle strength than for younger cyclists. We expected this impairment to be smaller if a pedelec was used. If such would be the case, this would indicate pedelecs to be beneficial for this older age group, because of a lower mental workload. Our study confirmed that increased physical effort in complex traffic sections deteriorated the detection of relevant stimuli in both age groups. Overall, older cyclists had longer reaction times and lower hit rates than younger cyclists. Mental workloads of cyclists are basically the same when cycling on a conventional bicycle or on a pedelec. In theory, pedelecs may be beneficial to reduce physical effort in cycling in order to maintain enough mental capacity to handle complex traffic situations. However, this study did not demonstrate these benefits. As pedelecs are often used for longer trips, by elderly with low muscle strength, future studies should also explore the effect of higher physical effort over longer periods of time, and also specifically in elderly with low muscle strength.

Genetic risk factors for clozapine-induced neutropenia and agranulocytosis in a Dutch psychiatric population.

Prescription of clozapine is complicated by the occurrence of clozapine-induced reduction of neutrophils. The aim of this study was to identify genetic risk factors in a population of 310 Dutch patients treated with clozapine, including 38 patients developing neutropenia and 31 patients developing agranulocytosis. NQO2 1541AA (NRH quinone oxidoreductase 2; protects cells against oxidative metabolites) was present at a higher frequency in agranulocytosis patients compared with control (23% versus 7%, P=0.03), as was ABCB1 (ABC-transporter-B1; drug efflux transporter) 3435TT (32% versus 20%, P=0.05). In patients developing neutropenia, ABCB1 3435TT and homozygosity for GSTT1null (glutathione-S-transferase; conjugates reactive clozapine metabolites into glutathione) were more frequent compared with control (34% versus 20%, P=0.05 and 31% versus 14%, P=0.03), whereas GSTM1null was less frequent in these patients (31% versus 52%, P=0.03). To investigate whether combinations of the identified genetic risk factors have a higher predictive value, should be confirmed in a larger case-control study.

Assessment of left ventricular ejection fraction in patients eligible for ICD therapy: Discrepancy between cardiac magnetic resonance imaging and 2D echocardiography.

OBJECTIVE: Implantable cardioverter defibrillators (ICD) and cardiac resynchronisation therapy (CRT) have substantially improved the survival of patients with cardiomyopathy. Eligibility for this therapy requires a left ventricular ejection fraction (LVEF) <35 %. This is largely based on studies using echocardiography. Cardiac magnetic resonance imaging (CMR) is increasingly utilised for LVEF assessment, but several studies have shown differences between LVEF assessed by CMR and echocardiography. The present study compared LVEF assessment by CMR and echocardiography in a heart failure population and evaluated effects on eligibility for device therapy. METHODS: 152 patients (106 male, mean age 65.5 ± 9.9 years) referred for device therapy were included. During evaluation of eligibility they underwent both CMR and echocardiographic LVEF assessment. CMR volumes were computed from a stack of short-axis images. Echocardiographic volumes were computed using Simpson's biplane method. RESULTS: The study population demonstrated an underestimation of end-diastolic volume (EDV) and end-systolic volume (ESV) by echocardiography of 71 ± 53 ml (mean ± SD) and 70 ± 49 ml, respectively. This resulted in an overestimation of LVEF of 6.6 ± 8.3 % by echocardiography compared with CMR (echocardiographic LVEF 31.5 ± 8.7 % and CMR LVEF 24.9 ± 9.6 %). 28 % of patients had opposing outcomes of eligibility for cardiac device therapy depending on the imaging modality used. CONCLUSION: We found EDV and ESV to be underestimated by echocardiography, and LVEF assessed by CMR to be significantly smaller than by echocardiography. Applying an LVEF cut-off value of 35 %, CMR would significantly increase the number of patients eligible for device implantation. Therefore, LVEF cut-off values might need reassessment when using CMR.

Active site substitution A82W improves the regioselectivity of steroid hydroxylation by cytochrome P450 BM3 mutants as rationalized by spin relaxation nuclear magnetic resonance studies.

Cytochrome P450 BM3 from Bacillus megaterium is a monooxygenase with great potential for biotechnological applications. In this paper, we present engineered drug-metabolizing P450 BM3 mutants as a novel tool for regioselective hydroxylation of steroids at position 16ß. In particular, we show that by replacing alanine at position 82 with a tryptophan in P450 BM3 mutants M01 and M11, the selectivity toward 16ß-hydroxylation for both testosterone and norethisterone was strongly increased. The A82W mutation led to a ≤42-fold increase in V(max) for 16ß-hydroxylation of these steroids. Moreover, this mutation improves the coupling efficiency of the enzyme, which might be explained by a more efficient exclusion of water from the active site. The substrate affinity for testosterone increased at least 9-fold in M11 with tryptophan at position 82. A change in the orientation of testosterone in the M11 A82W mutant as compared to the orientation in M11 was observed by T(1) paramagnetic relaxation nuclear magnetic resonance. Testosterone is oriented in M11 with both the A- and D-ring protons closest to the heme iron. Substituting alanine at position 82 with tryptophan results in increased A-ring proton-iron distances, consistent with the relative decrease in the level of A-ring hydroxylation at position 2ß.

Application of CYP102A1M11H as a tool for the generation of protein adducts of reactive drug metabolites.

Covalent binding of reactive metabolites (RMs) to proteins is considered to be one of the important mechanisms by which drugs can cause tissue damage. To facilitate the study of drug-protein adducts, we developed a potentially generic method for producing high levels of covalently modified proteins. A highly active drug metabolizing P450 BM3 mutant (CYP102A1M11H) is used for drug bioactivation. Because of its His-tag, CYP102A1M11H is easily removed by nickel affinity chromatography, facilitating subsequent characterization of the modified target protein. The applicability of our procedure is demonstrated by the trapping of RMs of acetaminophen (APAP), clozapine (CLOZ), and troglitazone (TGZ) with human glutathione-S-transferase P1-1 (hGST P1-1) as the model target protein. Tryptic digests of hGST P1-1 were subjected to analysis by LC-MS/MS and modified peptides identified by the comparative analysis of tryptic peptides of adducted and nonadducted hGST P1-1. Characteristic MS/MS ions of drug-modified peptides were identified by first searching for expected adduct-masses. Unanticipated drug-peptide adducts were subsequently identified in an unbiased manner by screening for diagnostic MS/MS ions of modified peptides. Reactive intermediates of APAP and CLOZ adducted to cysteine-47 and mass shifts corresponded to the alkylation of N-acetyl-p-benzoquinone imine (NAPQI) and the CLOZ nitrenium ion, respectively. Adduction of TGZ appeared more complex, yielding three different types of adducts to cysteine-47, two adducts to cysteine-14, and a single adduct to cysteine-101. Together, these findings show that P450 BM3 mutants with high capacity to activate drugs into relevant RMs can be employed to produce protein adducts to study the nucleophilic selectivity of highly reactive electrophiles.

Inhibition of human glutathione S-transferases by curcumin and analogues.

Glutathione S-transferases (GSTs) are important phase II drug-metabolizing enzymes that play a major role in protecting cells from the toxic insults of electrophilic compounds. Curcumin, a promising chemotherapeutic agent, inhibits human GSTA1-1, GSTM1-1, and GSTP1-1 isoenzymes. In the present study, the effect of three series of curcumin analogues, 2,6-dibenzylidenecyclohexanone (A series), 2,5-dibenzylidenecyclopentanone (B series), and 1,4-pentadiene-3-one (C series) substituted analogues (n = 34), on these three human GST isoenzymes, and on human and rat liver cytosolic GSTs, was investigated using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate. Most of the 34 curcumin analogues showed less potent inhibitory activities towards GSTA1-1, GSTM1-1, and GSTP1-1 than the parent curcumin. Compounds B14 and C10 were the most potent inhibitors of GSTA1-1 and human liver cytosolic GSTs, with IC(50) values of 0.2-0.6 microM. The most potent inhibitors of GSTM1-1 were C1, C3 and C10, with IC(50) values of 0.2-0.7 microM. Similarly, GSTP1-1 was predominantly strongly inhibited by compounds of the C series C0, C1, C2 C10 and A0, with IC(50) values of 0.4-4.6 microM. Compounds in the B series showed no significant inhibition of GSTP1-1. Molecular Operating Environment (MOE) program-based quantitative structure-activity relationship (QSAR) analyses have also suggested the relevance of Van der Waals surface area and compound lipophilicity factors for the inhibition of GSTA1-1 and GSTM1-1 and partial charge factors for GSTP1-1. These results may be useful in the design and synthesis of curcumin analogues with either more or less potency for GST inhibition.

Metabolism of N-substituted 7-methoxy-4-(aminomethyl) -coumarins by cytochrome P450 2D6 mutants and the indication of additional substrate interaction points.

Previous studies have shown the critical roles residues F120 and F483 play in the oxidative metabolism of 7-methoxy-4-(aminomethyl)-coumarin (MAMC) by cytochrome P450 2D6 (CYP2D6). In the present study, a series of N-alkyl-7-methoxy-4-(aminomethyl)-coumarins (MAMC analogues) were used as substrates for the F120A and F483A mutants in order to probe the CYP2D6 active site. The F120A and F483A mutants of CYP2D6 displayed significant activity towards the MAMC analogues. Automated docking studies of the MAMC analogues in a CYP2D6 homology model suggested a distal hydrophobic active site binding cleft for the substrate N-alkyl chains, consisting of the residues L213 and V308.

Bioactivation of N-substituted N'-(4-imidazole-ethyl)thioureas by human FMO1 and FMO3.

Enzyme kinetic parameters of the bioactivation of thiourea-containing compounds by human flavin-containing monooxygenase enzymes (FMOs) FMO1 and FMO3 were investigated. A microtitre-based adaptation of methodology described for the thiourea-dependent oxidation of thiocholine was used to determine the turnover of thiourea-containing compounds by human FMO1 and FMO3. The results show that major differences in enzyme kinetic parameters for N-substituted N'-(4-imidazole-ethyl)thiourea exist between human FMO3 and human FMO1. Whereas Km values of N-substituted N'-(4-imidazole-ethyl)thioureas for human FMO3 are all in the millimolar range, the Km values for human FMO1 range from the low micromolar to the low millimolar range. Furthermore, among a series of N-p-phenyl-substituted N'-(4-imidazole-ethyl)thioureas an interesting structure-activity relationship is evident with both FMO1 and FMO3. Where the Km decreases with increasing electron-withdrawing capacity of the p-substituent in the case of FMO1, the opposite phenomenon may be the case with FMO3. The kcat values of the compounds were all comparable for FMO1, averaging 3.03 +/- 0.56 min-1, whereas more variation was found for FMO3 (3.71 +/- 2.01 min-1). Enzyme kinetic parameters Km and kcat/Km of human FMO1 for N-substituted N'-(4-imidazole-ethyl)thioureas show a high degree of correlation with the results obtained in rat liver microsomes, in which rat FMO1 is the most abundant form, whereas those of human FMO3 do not.

Uptake-toxicity relationships of a series of N-substituted N'-(4-imidazole-ethyl)thiourea in precision-cut rat liver slices.

A previous study showed that the cytotoxicity of a series of N-p-phenyl-substituted N'-(4-imidazole-ethyl)thiourea in precision-cut rat liver slices increased with increasing electron-withdrawing capacity of the p-substituent and may be related to the Vmax/Km values of bioactivation of the thiourea-moiety by hepatic flavin-containing monooxygenases (FMOs). However, differences in the uptake of xenobiotics into precision-cut liver slices can also have consequences for the rates of metabolism of xenobiotics. In the present study, therefore, we investigated the rate and nature of uptake of 9 N-substituted N'-(4-imidazole-ethyl)thiourea into precision-cut rat liver slices. It was found that a five-fold difference exists among a series of N-substituted N'-(4-imidazole-ethyl)thiourea both in the initial rate of uptake and in the steady-state levels ultimately achieved in the precision-cut rat liver slices. It appeared that the most cytotoxic compounds were also the most readily absorbed compounds. The concentration-dependent initial rate of uptake could be described by a carrier-mediated saturable component and a non-saturable component. At cytotoxic concentrations, the non-saturable component accounted for more than 95% of the total uptake. From this study, it is concluded that differences in rate of uptake of thiourea-containing compounds may be a contributing factor to the differences in bioactivation by FMOs as the basis of the structure-toxicity relationships observed in precision-cut rat liver slices.

Use of 19F-nuclear magnetic resonance and gas chromatography-electron capture detection in the quantitative analysis of fluorine-containing metabolites in urine of sevoflurane-anaesthetized patients.

1: The use of fluorine-19 nuclear magnetic resonance (19F-NMR) and gas chromatography-electron capture detection (GC-ECD) in the analysis of fluorine-containing products in the urine of sevoflurane-exposed patients was explored. 2: Ten patients were anaesthetized by sevoflurane for 135-660 min at a flow rate of 6 l min(-1). Urine samples were collected before, directly after and 24 h after discontinuation of anaesthesia. 3: 19F-NMR analysis of the urines showed the presence of several fluorine-containing metabolites. The main oxidative metabolite, hexafluoroisopropanol (HFIP)-glucuronide, showed two strong quartet signals in the 19F-NMR spectrum. HFIP concentrations after beta-glucuronidase treatment were quantified by (19)F-nuclear magnetic resonance. Concentrations directly after and 24 h after discontinuation of anaesthesia were 131 +/- 41 (mean +/- SEM) and 61 +/- 19 mol mg(-1) creatinine, respectively. Urinary HFIP excretions correlated with sevoflurane exposure. 4: Longer scanning times enabled the measurement of signals from two compound A-derived metabolites, i.e. compound A mercapturic acid I (CAMA-I) and compound A mercapturic acid II (CAMA-II), as well as products from beta-lyase activation of the respective cysteine conjugates of compound A. The signals of the mercapturic acids, 3,3,3-trifluoro-2-(fluoromethoxy)-propanoic acid and 3,3,3-trifluorolactic acid were visible after combining and concentrating the patient urines. CAMA-I and -II excretions in patients were completed after 24 h. 5: Since 19F-nuclear magnetic resonance is not sensitive enough, urinary mercapturic acids concentrations were quantified by gas chromatography-electron capture detection. CAMA-I and -II urinary concentrations were 2.3 +/- 0.7 and 1.4 +/- 0.4 mol mg(-1) creatinine, respectively. Urinary excretion of CAMA-I showed a correlation with sevoflurane exposure, whereas CAMA-II did not. 6. The results show that 19F-nuclear magnetic resonance is a very selective and convenient technique to detect and quantify HFIP in non-concentrated human urine. 19F-nuclear magnetic resonance can also be used to monitor the oxidative biotransformation of sevoflurane in anaesthetized patients. Compound A-derived mercapturic acids and 3,3,3-trifluoro-2-(fluoromethoxy)-propanoic acid and 3,3,3-trifluorolactic acid, however, require more sensitive techniques such as gas chromatography-electron capture detection and/or gas chromatography-mass spectrometry for quantification.

Consensus molecular alignment based on generalized procrustes analysis.

One of the most serious problems in three-dimensional quantitative structure-activity relationship (3D-QSAR) studies is selection of an alignment rule for molecular super position of the compounds in the data set. In 3D-QSAR analyses of structure-activity data, a reference compound in a defined conformation is chosen, and all structures in the data set are aligned with the reference in a pairwise manner. In subsequent steps, conformation/alignment-dependent descriptors are computed for the compounds and compared to those of the reference. This approach gives much weight to the arbitrarily chosen reference molecule and can introduce a bias in the results. Here an alternative, and more general, approach to molecular alignment is presented that is based on Generalized Procrustes Analysis (GPA). The result is a consensus alignment that uses all molecules in the data set and avoids the bias introduced in the pairwise alignment strategy.

Studies on the inhibition of human cytochromes P450 by selenocysteine Se-conjugates.

1. To investigate whether cytochrome P450 (P450) inhibition can contribute to the chemopreventive activity of selenocysteine Se-conjugates (SeCys conjugates), 21 SeCys conjugates were screened for their inhibitory potency towards seven of the most important human P450s. 2. The majority of the SeCys conjugates produced near complete inhibition of CYP1A1 at a concentration of 250 microm. The most potent inhibitor, Se-benzyl-L-selenocysteine, displayed an IC50 of 12.8 +/- 1.2 microm. CYP2C9, -2C19 and -2D6 were moderately (50-60%) inhibited by the SeCys conjugates. CYP1A2, -2E1 and -3A4 were least inhibited. 3. Studies on the susceptibility of CYP1A1 to SeCys conjugates implicated a thiol-reactive intermediate, as evidenced by reduced inhibition levels in the presence of glutathione and N-acetyl cysteine. Uncoupling of the P450-catalytic cycle was of no importance as ROS scavengers did not influence inhibition levels. 4. P450 inhibition by two physiologically relevant metabolite classes of SeCys conjugates was also studied. N-acetylation of SeCys conjugates consistently increased the inhibitory potency towards CYP1A2, -2C19, -2E1 and -3A4. Beta-lyase catalysed bioactivation of alkyl-substituted SeCys conjugates or Se-benzyl-L-selenocysteine produced little or no additional inhibition of P450 activity. For Se-phenyl-L-selenocysteine, however, significant increases in P450 inhibition were obtained by beta-lyase pre-incubation. 5. It is concluded that the potent and relatively selective CYP1A1 inhibition exerted by SeCys conjugates may contribute to their chemopreventive activity.

Midazolam is a phenobarbital-like cytochrome p450 inducer in rats.

Midazolam is almost exclusively metabolized by cytochrome P450 3A (CYP3A) isoenzymes. Therefore, midazolam is used as a probe to determine CYP3A levels in humans and rats. A prerequisite for longitudinal determination of CYP3A expression levels using midazolam as a probe is that midazolam itself has no effect on the expression of CYP3A. In the present study, we analyzed the mRNA levels and enzyme activities of the major CYP isoforms in the rat liver after intraperitoneal injection of midazolam (50 mg/kg) for 3 consecutive days. CYP3A1 mRNA levels were increased 4-fold in midazolam-treated animals compared with controls, whereas the mRNA levels of CYP3A2, CYP3A9, and CYP3A18 were not altered. The increase in CYP3A1 mRNA was accompanied by a 25% increase in microsomal testosterone 6beta-hydroxylation activity. More strikingly, CYP2B1/2 mRNA levels were increased 22-fold upon midazolam treatment, leading to an 11- to 95-fold enhancement of CYP2B enzyme activity. CYP2C6 mRNA levels were 4 times higher in midazolam-treated animals. Formation of 2alpha-hydroxy-testosterone, mainly catalyzed by CYP2C11, was 2.6-fold lower in liver microsomes from midazolam-treated animals. Midazolam induced CYP2E enzyme activity 2.5-fold at the post-transcriptional level. The induction of CYP2B1/2 mRNA levels by midazolam was dose-dependent (4.5-fold increase at 10 mg/kg). Induction of CYP3A1 and CYP2B expression was also observed in isolated rat hepatocytes cultured with 100 microM midazolam. We conclude that midazolam is a phenobarbital-like CYP inducer in rats. Induction of CYP3A1 by midazolam may have implications for the longitudinal use of midazolam as a probe for analysis of CYP3A expression levels in rats.

Bioactivation of chemopreventive selenocysteine Se-conjugates and related amino acids by amino acid oxidases novel route of metabolism of selenoamino acids.

Several selenocysteine Se-conjugates have been shown to possess potent chemopreventive activity in animal models for chemical carcinogenesis. As a mechanism of action, beta-elimination reactions to form chemopreventive selenols, ammonia, and pyruvate has been proposed. The enzymes involved in these beta-elimination reactions, however, have been partially elucidated. Next to cysteine conjugate beta-lyases, as yet unidentified non-pyridoxal-5'-phosphate-dependent enzymes also appear to be involved in cytosolic beta-elimination reactions. In the present study, it was investigated whether amino acid oxidases contribute to the bioactivation of selenocysteine Se-conjugates. Using purified L-amino acid oxidase from Crotalus adamanteus as a model enzyme, significant beta-elimination activities were indeed observed upon incubation with Se-methylselenocysteine (K(m), 195 microM; k(cat), 48 min(-1)), Se-allylselenocysteine (K(m), 608 microM; k(cat), 34 min(-1)), Se-phenylselenocysteine (K(m), 107 microM; k(cat), 57 min(-1)) and Se-benzylselenocysteine (K(m), 59 microM; k(cat), 13 min(-1)). For all selenocysteine Se-conjugates tested, the rate of pyruvate formation was comparable to that of hydrogen peroxide, one of the products of oxidative deamination. The fact that addition of catalase did not alter pyruvate formation indicated that the beta-elimination reaction observed was not mediated by selenoxidation/syn-elimination due to the hydrogen peroxide formed via the oxidative deamination pathway. Using D-amino acid oxidase from porcine kidney and D-SeCys conjugates similar results were obtained. To delineate whether mammalian L-amino acid oxidases are also able to catalyze beta-elimination of selenocysteine Se-conjugates, rat renal cytosol was fractionated and screened for beta-elimination and oxidative deamination activities. One of the fractions isolated displayed oxidative deamination activity with several amino acids and cysteine S-conjugates. With selenocysteine Se-conjugates as substrates, however, this fraction displayed both oxidative deamination and beta-elimination activities, when incubated in the presence of aminoxyacetic acid to block contribution of pyridoxal-5'-phosphate-dependent enzymes. The potential significance of this novel bioactivation route for the chemopreventive activity of selenocysteine Se-conjugates is discussed.

Characterization of enzymes involved in biotransformation of polycyclic aromatic hydrocarbons in terrestrial isopods.

Little is known about the capacity of terrestrial invertebrates to transform organic soil pollutants such as polycyclic aromatic hydrocarbons (PAHs). Studies were designed to characterize microsomal mixed function oxygenase and accompanying conjugation enzymes from the hepatopancreas of the terrestrial isopods Porcellio scaber and Oniscus asellus using pyrene and 1-hydroxypyrene as model substrates. The hydroxylation of pyrene and the formation of pyreneglucoside and pyrenesulfate appeared to be sensitive measures for the activity of cytochrome P450 aryl hydrocarbon hydroxylase (AHH), uridinediphosphateglucosyltransferase (UDPGT), and aryl sulfotransferase (ST), respectively. Treatment with the antibiotic riphampicine demonstrated that the enzyme activities originate from the animals themselves and not from symbiotic microflora present in the hepatopancreas and the gut. In both species, ST has a very high affinity for 1-hydroxypyrene with Km values two orders of magnitude lower than that of UDPGT. The Vmax values of UDPGT, however, are 10- to 20-fold higher than that of ST. Taking the P450 activities into consideration, both species are expected to transform PAHs in an equally effective way. When the isopods were fed with food containing benz[a]pyrene and 3-methyl-cholanthrene, none of the enzyme activities appeared to be inducible except for a small enhancement of UDPGT in O. asellus. Our findings indicate that terrestrial isopods have a high, noninducible capacity for biotransformation of PAHs and that the sulfate conjugation pathway is as important as the carbohydrate conjugation pathway. This conclusion is consistent with the low body residues of parent PAHs found in the field.

Characterization of thioether compounds formed from alkaline degradation products of enflurane.

BACKGROUND: Renal toxicity has occasionally been observed after enflurane anesthesia. Although originally attributed to its oxidative metabolism to inorganic fluoride, serum levels of inorganic fluoride appear to be small to explain these renal effects. Formation of potentially nephrotoxic halogenated alkenes during alkaline degradation in carbon dioxide absorbers and subsequent bioactivation via the glutathione conjugation pathway may be considered as an alternative mechanism for renal toxicity. The aim of this study was to characterize the thioethers formed chemically and biosynthetically. METHODS: Alkaline degradation of enflurane was achieved by stirring with pulverized potassium hydroxide. Volatile degradation products were analyzed by 19F nuclear magnetic resonance (NMR) analysis of head space gasses trapped in dimethyl sulfoxide (DMSO). Thioethers were generated chemically by trapping head space gasses in DMSO containing N-acetyl-L-cysteine or 2-mercaptoacetic acid as model thiol compounds. Glutathione conjugates were generated biosynthetically by passing head space through rat liver fractions in presence of glutathione. Products formed were analyzed by gas chromatography-mass spectroscopy and 19F-NMR. RESULTS: Direct analysis of head space gasses showed formation of 1-chloro-1,2-difluorovinyl difluoromethyl ether and two unidentified fluorine-containing products as alkaline degradation products of enflurane. When trapped in DMSO-N-acetyl-L-cysteine-triethylamine, N-acetyl-S-(2-chloro-1,2-difluoro-1-(difluoromethoxy)ethyl)-L-cysteine was identified as the major product. Another N-acetyl-L-cysteine S-conjugate formed was N-acetyl-S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine, a potent nephrotoxin in rats. 19F-NMR analysis of glutathione conjugates formed after incubation with rat liver fractions resulted in formation of corresponding S-conjugates. CONCLUSIONS: The current study demonstrates that alkaline degradation products of enflurane can be conjugated to thiol compounds, forming S-conjugates that could theoretically contribute to adverse renal effects observed occasionally with enflurane anesthesia. The N-acetyl-L-cysteine S-conjugates identified may be biomarkers to assess exposure of humans to alkaline degradation products of enflurane.

Study on the cytochrome P-450- and glutathione-dependent biotransformation of trichloroethylene in humans.

OBJECTIVES: To investigate in humans the contribution of the cytochrome P-450- and glutathione-dependent biotransformation of trichloroethylene (TRI) under controlled repeated exposure in volunteers, and under occupational conditions. METHODS: Volunteers were exposed to TRI, using repeated 15 min exposures at 50 and 100 ppm. This exposure schedule resulted in internal doses of 1.30 and 2.40 mmol of TRI respectively. Urine samples were collected for a minimum of 45 h. Urine samples were also collected from occupationally exposed workers. The samples were analysed for the known human metabolites of TRI, trichloroethanol (TCE), trichloroacetic acid (TCA) and both regio-isomeric forms of the mercapturic acid N-acetyl-S-(dichlorovinyl)-L-cysteine (DCV-NAC), and for (dichlorovinyl)-L-cysteine (DCVC). In order to further elucidate the metabolism of TRI in humans, we analysed samples for dichloroacetic acid and for the proposed break-down products of 1,2 and 2,2-dichlorovinyl-L-cysteine after deamination: the S-conjugates of 3-mercaptolactic acid, 3-mercaptopyruvic acid and 2-mercaptoacetic acid. RESULTS: None of the glutathione metabolites was found in urine of volunteers. In workers occupationally exposed to TRI at levels between 0.4 and 21 ppm [8-h time-weighted average (TWA)], levels of DCV-NAC in urine samples collected at the end of the 4th working day and also next morning were below detection limit (0.04 mumol/l). This confirms the findings of Bernauer et al. (1996) that these metabolites are excreted at very low levels in humans. Urinary levels of DCVC and six postulated metabolites of dichlorovinyl-S-cysteine conjugates via deamination were also below 0.04 mumol/l, indicating that at most 0.05% of the dose is excreted in the form of these metabolites. These data further strengthen the argument for a very low activity of glutathione-mediated metabolism for chronically exposed workers. CONCLUSIONS: This study gives additional data which indicate that glutathione-mediated metabolism is of minor importance in humans exposed to TRI. In spite of indications to the contrary, significant metabolism of the cysteine conjugate via beta-lyase, which could result in a toxic metabolite, cannot be ruled out completely.

Characterization of biotransformation enzyme activities in primary rat proximal tubular cells.

The proximal tubule is a frequent target for nephrotoxic compounds due to it's ability to transport and accumulate xenobiotics and their metabolites, as well as by the presence of an organ-selective set of biotransformation enzymes. The aim of the present study was to characterize the activities of different biotransformation enzymes during primary culturing of rat proximal tubular cells (PT cells). Specific marker substrates for determining cytochrome P450 (CYP450) activity of primary cultured PT cells include 7-ethoxyresorufin (CYP1A1), caffeine (CYP1A), testosterone (CY2B/C, CYP3A), tolbutamide (CYP2C) and dextromethorphan (CYP2D1). Activities of the CYP450 isoenzymes decreased considerably during culture with the greatest loss in activity within 24 h of culture. In addition, expression of CYP450 apoprotein, including CYP1A, CYP2C, CYP2D, CYP2E and CYP4A, was detected in microsomes from freshly isolated PT cells by immunoblotting using specific antibodies. CYP2B and CYP3A apoprotein could not be detected. Activity of the phase II biotransformation enzymes GST, GGT, beta-lyase and UGT was determined with 1-chloro-2,4-dinitrobenzene, L-glutamic acid gamma-(7-amido-4-methyl-coumarin), S-(1,1,2,2-tetrafluoroethyl)-L-cysteine and 1-naphthol, respectively, as marker substrates. Activity of the phase II enzymes remained more stable and, in contrast to CYP450 activity, significant activity was still expressed after 1 week of PT cell culture. Thus, despite the obvious advantages of PT cells as an in-vitro model for studies of biotransformation mediated toxicity, the strong time dependency of especially phase I and, to a lesser extent, phase II biotransformation activities confers limitations to their application.