Glucose metabolism and hemoglobin reactivity in human red blood cells exposed to the tryptophan metabolites 3-hydroxyanthranilate, quinolinate and picolinate

Glucose metabolism and hemoglobin reactivity in intact human erythrocytes were assessed in the presence of the tryptophan metabolites, 3-hydroxyanthranilate (3-HAT), quinolinate and picolinate. Of these compounds, only 3-HAT altered red cell oxidative status by inducing, in a dose-dependent manner, formation of methemoglobin and non-functional oxidation products of hemoglobin, and by increasing both net glycolytic flux and flux through the hexose monophosphate shunt. 3-HAT also decreased the normal lactate to pyruvate production ratio with pyruvate accumulating at the expense of lactate. These findings are consistent with the auto-oxidative reactivity of quinolinate, picolinate, and 3-HAT in that only 3-HAT undergoes base-catalyzed auto-oxidation (Dykens et al., Biochem Pharmacol 36: 211-217, 1987). Lactate and pyruvate added to the medium in physiologic concentrations uncoupled oxidative glycolysis from reductive glycolysis, resulting in accumulation of pyruvate in the presence of 3-HAT with little increase in total glycolytic flux. Superoxide dismutase (SOD), which accelerates 3-HAT auto-oxidation in vitro (Dykens et al., Biochem Pharmacol 36: 211-217, 1987), exacerbated HAT-mediated oxidative insult by increasing methemoglobin formation, hexose monophosphate shunt flux, and pyruvate accumulation. Persistence of 3-HAT-induced red cell metabolic responses and oxidative damage in the presence of SOD, DETAPAC (diethylenetriaminepentaacetic acid) and formate suggests that an organic-based radical, perhaps the anthranilyl radical produced during 3-HAT auto-oxidation, is the proximate agent exerting oxidative stress. Slow rates of auto-oxidation indicate that 3-HAT may be useful as a probe of antioxidant mechanisms in normal and diseased red blood cells.     

13C(2)-Labeled methyl tert-butyl ether: toxicokinetics and characterization of urinary metabolites in humans.

After exposure to methyl tert-butyl ether (MTBE), a gasoline additive, only one metabolite [tert-butyl alcohol (TBA), <1% of dose] has been identified in human urine [Nihlén, A., et al. (1998) Toxicol. Appl. Pharmacol. 148, 274-280]. In the study presented here, metabolites of MTBE were characterized by (1)H-decoupled (13)C NMR spectroscopy in urine obtained from four volunteers experimentally exposed to 50 ppm (13)C-labeled MTBE ([1,2-(13)C(2)]MTBE) vapor (facemask) for 2 h during a light physical work load (50 W). Chemical shifts for the two adjacent (13)C-labeled carbons in [1, 2-(13)C(2)]MTBE-derived metabolites were consistent with the shifts obtained for spiked standards of alpha-hydroxyisobutyric acid (HBA) and 2-methyl-1,2-propanediol (MPD). NMR signals were not detected for labeled MTBE, TBA, or possible MTBE-derived conjugates. Quantification of HBA and MPD was performed by NMR for two urine samples (collected 20 h after exposure). One subject had 11% HBA and 1% MPD, and the other individual had 3% HBA and 1% MPD in the urine, expressed as a percentage of MTBE uptake. This indicates that HBA and MPD occur at significantly higher levels in the urine (detected by NMR) than MTBE and TBA (detected by GC). To our knowledge, this is the first characterization of MTBE metabolites, other than TBA, in humans. Further urine, blood, and expired air were collected up to 22 h after exposure, and the toxicokinetics of MTBE, TBA, and acetone were determined by GC. Low relative uptake (39%), a low level of postexposure exhalation of MTBE (17%), and low recovery of TBA in the urine (<1%) were observed. The same subjects had previously been exposed to unlabeled MTBE in a whole-body exposure study [Nihlén, A., et al. (1998) Toxicol. Appl. Pharmacol. 148, 274-280], and the toxicokinetics of MTBE and TBA in this facemask exposure did not differ from the previous whole-body chamber exposure.     

Cytogenetic biomarkers, urinary metabolites and metabolic gene polymorphisms in workers exposed to styrene

The present study comprised a biomonitoring study in 95 workers occupationally exposed to styrene and 98 unexposed controls, employing an integrated approach involving biomarkers of exposure, effect, and susceptibility. Airborne styrene was evaluated at workplace, and urinary styrene metabolites, mandelic acid (MA), phenylglyoxylic acid (PGA), vinylphenols (VPTs) and phenylhydroxyethylmercapturic acids (PHEMAs), were measured as biomarkers of internal dose. Cytogenetic alterations were evaluated by analysing the frequency of chromosomal aberrations (CAs) and micronucleated binucleated cells (MNBN) in peripheral blood lymphocytes. The micronucleus assay was coupled with centromeric fluorescence in situ hybridization to distinguish micronuclei (MN) arising from chromosomal breakage (C- MN) from those harboring whole chromosomes (C+ MN). The possible influence of genetic polymorphisms of xenobiotic-metabolizing enzymes involved in styrene biotransformation (EPHX1, GSTT1, GSTM1, GSTP1) and NAT2 on the cytogenetic endpoints was investigated. The exposed workers showed a significantly higher frequency of MNBN (13.8+/-0.5% versus 9.2+/-0.4%; P<0.001) compared to control subjects. The effect appeared to concern both C- and C+ MN. A positive correlation was seen between the frequency of C+ MN and urinary level of MA+PGA (P<0.05) and VPTs (P<0.001). Chromosome-type CAs positively correlated with airborne styrene level and VPTs (P<0.05), whereas chromatid-type CAs correlated with PHEMAs (P<0.05). Workers bearing GSTM1 null genotype showed lowered levels of PHEMAs (P<0.001). The GSTT1 null genotype was associated with increased MNBN frequencies in the exposed workers (P<0.05) and the fast activity EPHX genotype with a moderate decrease in both MNBN and CAs in the controls. Our results suggest that occupational exposure to styrene has genotoxic effects that are potentiated by the GSTT1 gene deletion. These observations may have relevance considering the risk of lymphatic and haematopoietic malignancies tentatively associated with styrene exposure.     

Analysis of hemoglobin adducts from acrylamide, glycidamide, and ethylene oxide in paired mother/cord blood samples from Denmark

The knowledge about fetal exposure to acrylamide/glycidamide from the maternal exposure through food is limited. Acrylamide, glycidamide, and ethylene oxide are electrophiles and form adducts with hemoglobin (Hb), which could be used for in vivo dose measurement. In this study, a method for analysis of Hb adducts by liquid chromatography-mass spectrometry, the adduct FIRE procedure, was applied to measurements of adducts from these compounds in maternal blood samples (n = 87) and umbilical cord blood samples (n = 219). The adduct levels from the three compounds, acrylamide, glycidamide, and ethylene oxide, were increased in tobacco smokers. Highly significant correlations were found between cord and maternal blood with regard to measured adduct levels of the three compounds. The mean cord/maternal hemoglobin adduct level ratios were 0.48 (range 0.27-0.86) for acrylamide, 0.38 (range 0.20-0.73) for glycidamide, and 0.43 (range 0.17-1.34) for ethylene oxide. In vitro studies with acrylamide and glycidamide showed a lower (0.38-0.48) rate of adduct formation with Hb in cord blood than with Hb in maternal blood, which is compatible with the structural differences in fetal and adult Hb. Together, these results indicate a similar life span of fetal and maternal erythrocytes. The results showed that the in vivo dose in fetal and maternal blood is about the same and that the placenta gives negligible protection of the fetus to exposure from the investigated compounds. A trend of higher levels of the measured adducts in cord blood with gestational age was observed, which may reflect the gestational age-related change of the cord blood Hb composition toward a higher content of adult Hb. The results suggest that the Hb adduct levels measured in cord blood reflect the exposure to the fetus during the third trimester. The evaluation of the new analytical method showed that it is suitable for monitoring of background exposures of the investigated electrophilic compounds in large population studies.     

Benzo(a)pyrenediolepoxide-hemoglobin adducts and 3-hydroxy-benzo(a)pyrene urinary excretion profiles in rats subchronically exposed to benzo(a)pyrene

 The time profiles of benzo(a)pyrenediolepoxide (BaPDE)-hemoglobin (Hb) adduct formation and 3-hydroxybenzo(a)pyrene (3-OHBaP) urinary excretion were studied in male Sprague-Dawley rats exposed to daily benzo(a)pyrene (BaP) intraperitoneal doses of 1.25, 6.25, and 31.25 μmol/kg administered Tuesday to Friday for 4 consecutive weeks. Blood was withdrawn weekly, on Tuesdays, prior to dosing. Twenty four hour urine samples were collected on Mondays (following 72 h without treatment) and Thursdays. Analytes were quantified by high performance liquid chromatography (HPLC)/fluorescence. Exposure to BaP resulted in the accumulation of BaPDE-Hb adducts, reaching an average of 1.2±0.3, 8.3±1.9, and 38.2±6.1 pmol/g Hb for the 1.25, 6.25, and 31.25 μmol/kg per day doses after 4 weeks of treatment. The expected saw tooth excretion profile of 3-OHBaP was observed, with peaks on Thursdays and troughs on Mondays, and showed a progressive rise on both Mondays and Thursdays. Increase in Monday values with time suggested a possible increase in BaP body burden during exposure. To verify this aspect further, the urinary excretion kinetic of 3-OHBaP following acute intraperitoneal dosing (31.25 μmol/kg) was determined. Urine samples were collected at frequent timed intervals for up to 164 h post-dosing. Two-step elimination was observed, the second step having a half-life of 25 h, presumably linked to the slow release of BaP accumulated in fatty tissues upon repeated treatment. Therefore, it seems that 1) BaPDE-Hb adducts are good indicators of repeated exposure, 2) the difference between pre- and post-exposure 3-OHBaP urinary excretion gives a measure of recent exposure, and 3) excretion levels of this latter metabolite after a non-exposure period of sufficient duration, to allow elimination of the bulk BaP from the last dose, could reflect BaP body burden. Received: 3 November 1994/Accepted: 11 January 1995     

Proteomic characterization of metabolites, protein adducts, and biliary proteins in rats exposed to 1,1-dichloroethylene or diclofenac

A proteome profiling approach was used to compare effects of two toxicants, 1,1-dicloroethylene (DCE) and diclofenac, which covalently adduct hepatic proteins. Bile was examined as a potential source of protein alterations since both toxicants target the hepatic biliary canaliculus. Bile was collected before and after toxicant treatment. Biliary proteins were separated by one-dimensional SDS-PAGE and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS-MS) with data-dependent scanning. Comprehensive analysis of biliary proteins was performed by using SEQUEST and BLAST database searching, in combination with de novo interpretation. Bile not subjected to tryptic digestion was analyzed for DCE metabolites. DCE treatment resulted in a marked increase in the overall number of biliary proteins, whereas few changes in the proteomic profile were apparent in bile after diclofenac treatment. This is consonant with prior observations of more profound effects of DCE on canalicular membrane integrity. LC-MS-MS analyses for DCE metabolites revealed the presence of S-carboxymethyl glutathione, S-(cysteinylacetyl)glutathione, and a product of the intramolecular rearrangement of the DCE metabolite, ClCH(2)COSG, not previously described in vivo. In addition, several S-carboxymethylated proteins were identified in bile from DCE-treated animals. This investigation has produced the first comprehensive baseline characterization of the content of the rat biliary proteome and the first documentation of alterations in the proteome of bile by toxicant treatment. In addition, the results provide direct in vivo evidence for DCE metabolic routes proposed in the formation of covalent adducts.     

Effect of ascorbic acid supplementation on nitric oxide metabolites and systolic blood pressure in rats exposed to lead

Background:

Extended exposure to low levels of lead causes high blood pressure in human and laboratory animals. The mechanism is not completely recognized, but it is relatively implicated with generation of free radicals, oxidant agents such as ROS, and decrease of available nitric oxide (NO). In this study, we have demonstrated the effect of ascorbic acid as an antioxidant on nitric oxide metabolites and systolic blood pressure in rats exposed to low levels of lead.

Materials and Methods:

The adult male Wistar rats weighing 200-250 g were divided into four groups: control, lead acetate (receiving 100 ppm lead acetate in drinking water), lead acetate plus ascorbic acid (receiving 100 ppm lead acetate and 1 g/l ascorbic acid in drinking water), and ascorbic acid (receiving 1 g/l ascorbic acid in drinking water) groups. The animals were anesthetized with ketamin/xylazine (50 and 7 mg/kg, respectively, ip) and systolic blood pressure was then measured from the tail of the animals by a sphygmomanometer. Nitric oxide levels in serum were measured indirectly by evaluation of its stable metabolites (total nitrite and nitrate (NOχ)).

Results:

After 8 and 12 weeks, systolic blood pressure in the lead acetate group was significantly elevated compared to the control group. Ascorbic acid supplementation could prevent the systolic blood pressure rise in the lead acetate plus ascorbic acid group and there was no significant difference relative to the control group. The serum NOχ levels in lead acetate group significantly decreased in relation to the control group, but this reduction was not significantly different between the lead acetate plus ascorbic acid group and the control group.

Conclusion:

Results of this study suggest that ascorbic acid as an antioxidant prevents the lead induced hypertension. This effect may be mediated by inhibition of NOχ oxidation and thereby increasing availability of NO.     

Rat tissue and blood partition coefficients for n-alkanes (C8 to C12)

Rat tissue:air and blood:air partition coefficients (PCs) for octane, nonane, decane, undecane, and dodecane (n-C8 to n-C12 n-alkanes) were determined by vial equilibration. The blood:air PC values for n-C8 to n-C12 were 3.1, 5.8, 8.1, 20.4, and 24.6, respectively. The lipid solubility of n-alkanes increases with carbon length, suggesting that lipid solubility is an important determinant in describing n-alkane blood:air PC values. The muscle:blood, liver: blood, brain:blood, and fat:blood PC values were octane (1.0, 1.9, 1.4, and 247), nonane (0.8, 1.9, 3.8, and 274), decane (0.9, 2.0, 4.8, and 328), undecane (0.7, 1.5, 1.7, and 529), and dodecane (1.2, 1.9, 19.8, and 671), respectively. The tissue:blood PC values were greatest in fat and the least in muscle. The brain:air PC value for undecane was inconsistent with other n-alkane values. Using the measured partition coefficient values of these n-alkanes, linear regression was used to predict tissue (except brain) and blood:air partition coefficient values for larger n-alkanes, tridecane, tetradecane, pentadecane, hexadecane, and heptadecane (n-C13 to n-C17). Good agreement between measured and predicted tissue:air and blood:air partition coefficient values for n-C8 to n-Cl2 offer confidence in the partition coefficient predictions for longer chain n-alkanes.     

Characterization of urinary metabolites from [1,2,methoxy-13C]-2-methoxyethanol in mice using 13C nuclear magnetic resonance spectroscopy.

2-Methoxyethanol (2-ME) is an industrial solvent that induces developmental and testicular toxicity in laboratory animals. Oxidation of 2-ME to 2-methoxyacetic acid (2-MAA) is required for the generation of these adverse effects. The urinary metabolites of 2-ME were investigated to characterize the fate of 2-ME and 2-MAA. 13C NMR spectroscopy was used to detect and assign metabolites in the urine of pregnant CD-1 mice following administration of 250 mg/kg of [1,2,methoxy-13C]-2-ME. Two-dimensional NMR methods were used to correlate signals from the labeled carbons in each 2-ME metabolite and to determine the number of hydrogens attached to each carbon. Structures were assigned from the NMR data together with calculated values of shift for biochemically feasible metabolites and by comparison to standards. Pathways involved in forming metabolites assigned in this study include transformation of 2-ME via ethylene glycol, conjugation with glucuronide or sulfate, and oxidation to 2-MAA. Additional metabolites were assigned that can be formed from further conversion of 2-MAA to glycine and glucuronide conjugates, as well as metabolites derived from the incorporation of 2-methoxyacetyl CoA derivatives into intermediary metabolism. Elucidation of the further metabolism of 2-MAA may be important for understanding the mechanisms by which 2-ME induces adverse effects.     

DNA adducts in liver and leukocytes of flounder (Platichthys flesus) experimentally exposed to benzo

In the present study the levels of hydrophobic DNA adducts detected by 32P-postlabelling were followed in liver and leukocytes of flounder (Platichthys flesus) over 10 days following single i.p. injections of two doses of BaP (10 and 50 mg kg(-1) fish weight, respectively). DNA adducts were detected in both tissues of exposed fish 2 days post injection and continued to rise on day 5 and day 10. In flounder exposed to the lower dose of BaP, the levels of hepatic DNA adducts reached higher values on the fifth day compared with flounder exposed to the higher dose. However, at the end of the experiment, the DNA adduct level was again higher in fish from the high dose group compared with the low dose group. There was no substantial increase of DNA adducts in liver of flounder from the low dose group after day 5, while the adduct levels in flounder liver from the high dose group increased throughout the experiment. Earlier studies detecting DNA adducts in BaP-exposed flatfish with the 32P-postlabelling technique have reported declining adduct levels from about 2 days after the exposure, regardless of exposure route. In contrast, the results from our study did not confirm a rapid increase and successive decline of hydrophobic adducts in liver of BaP-exposed flounder.     

应用HPLC同时测定大鼠尿中N,N-二(正丁基)阿霉素-14-戊酸酯及其8种代谢产物(英文)

目的,同时测定大鼠尿中N,N-二(正丁基)阿霉素-14-戊酸酯及其8种代谢产物 方法:建立了一种反相高压液相色谱法,大鼠iv 20mg·kg~(-1)原药后,其尿直接进样.梯度洗脱,荧光检测.结果:原药最低检出量2 ng,代谢物1—3 ng.被检物不受尿成分干扰.72 h尿中总葸环荧光信号仅为剂量的4.9％,其中主要为脱酰基以及N-脱丁基代谢物.6种次要代谢物包括苷元以及13—酮基还原性代谢物等,但未检出葡萄糖醛酸结合物.结论:本法简便易行,灵敏度高,特异性强。     

Interdependence of hemoglobin, catalase and the hexose monophosphate shunt in red blood cells exposed to oxidative agents

Hydrogen peroxide, 1, 4-naphthoquinone-2-sulfonic acid and 6-hydroxydopamine were used as biochemical probes to study the interdependence of hemoglobin, catalase and the hexose monophosphate shunt in protection of red blood cell (red cell) function against Superoxide, hydrogen peroxide and organic free radicals. It was shown that catalase may remove hydrogen peroxide both catalatically and peroxidatically in the red cell and that glucose metabolism supplies electron donors for the peroxidatic function of catalase. The hexose monophosphate shunt is known to participate in removal of hydrogen peroxide by supplying electrons for the action of glutathione reductase and glutathione peroxidase. Experiments in which red cell catalase was irreversibly inhibited by interaction with hydrogen peroxide and 3-amino-1, 2, 4-triazole showed that both catalase and the hexose monophosphate shunt share in the removal of hydrogen peroxide from red cells. The effect of the hemoglobin oxidation state on the interaction of red cells and oxidative agents was studied using red cell preparations containing hemoglobin, carbonmonoxyhemoglobin or methemoglobin. Oxyhemoglobin was able to accelerate the production of Superoxide and hydrogen peroxide with agents like 1, 4-naphthoquinone-2-sulfonic acid, whereas oxyhemoglobin had little effect with 6-hydroxydopamine. In experiments with red cells containing oxyhemoglobin, the accumulation of catalase in the form of Compound II, with resulting loss of available catalatic activity, was directly proportional to formation of methemoglobin. The close relationship between loss of catalatic activity and formation of methemoglobin may indicate that catalase has a protective effect on hemoglobin. Experiments with red cells containing methemoglobin indicated that once methemoglobin is formed it protects the red cell from further loss of the catalatic activity of catalase. In some circumstances oxyhemoglobin was formed from methemoglobin as a by-product of the protective effect of methemoglobin on red cell function. Formation of oxyhemoglobin by this mechanism was many times faster than oxyhemoglobin formation by the methemoglobin reductase system.     

Characterization and quantitation of urinary metabolites of [1,2,3-13C]acrylamide in rats and mice using 13C nuclear magnetic resonance spectroscopy.

Acrylamide, widely used for the production of polymers and as a grouting agent, causes neurotoxic effects in humans and neurotoxic, genotoxic, reproductive, and carcinogenic effects in laboratory animals. In this study, 13C NMR spectroscopy was used to detect metabolites of acrylamide directly in the urine of rats and mice following administration of [1,2,3-13C]acrylamide (50 mg/kg po). Two-dimensional NMR experiments were used to correlate carbon signals for each metabolite in the urine samples and to determine the number of hydrogens attached to each carbon. Metabolite structures were identified from the NMR data together with calculated values of shift for biochemically feasible metabolites and by comparison with standards. The metabolites assigned in rat and mouse urine are N-acetyl-S-(3-amino-3-oxopropyl)cysteine, N-acetyl-S-(3-amino-2-hydroxy-3-oxopropyl)cysteine, N-acetyl-S-(1-carbamoyl-2-hydroxy-ethyl)cysteine, glycidamide, and 2,3-dihydroxypropionamide. These metabolites arise from direct conjugation of acrylamide with glutathione or from oxidation to the epoxide, glycidamide, and further metabolism. Acrylamide was also detected in the urine. Quantitation was carried out by integrating the metabolite carbon signals with respect to that of dioxane added at a known concentration. The major metabolite for both the rat (70% of total metabolites excreted) and the mouse (40%) was formed from direct conjugation of acrylamide with glutathione. The remaining metabolites for the rat (30%) and mouse (60%) are derived from glycidamide. The species differences in extent of metabolism through glycidamide may have important consequences for the toxic and carcinogenic effects of acrylamide.     

Elimination of cocaine and metabolites in plasma, saliva, and urine following repeated oral administration to human volunteers

Chronic administration of lipophilic drugs can result in accumulation and prolonged elimination during abstinence. It has been suggested that cocaine and/or metabolites can be detected in saliva and urine for an extended period following long-term, high-dose administration. The effects of chronic oral cocaine administration in healthy volunteer subjects with a history of cocaine abuse were investigated. Subjects were housed on a closed clinical ward and were administered oral cocaine in up to 16 daily sessions. In each session, volunteers received five equal doses of oral cocaine with 1 h between doses. Across sessions, cocaine was administered in ascending doses from an initial dose of 100 mg (500 mg/day) up to 400 mg (2 g/day), increasing by 25 mg/dose/session (125 mg/session). Participation in the study was terminated if cardiovascular safety parameters were exceeded. Plasma and saliva specimens were collected periodically during the dosing sessions and during the one-week withdrawal phase at the end of the study. All urine specimens were collected throughout the entire study. Specimens were analyzed for cocaine and metabolites by solid-phase extraction followed by gas chromatographic-mass spectrometric analysis in the SIM mode. The limit of detection for each analyte was approximately 1 ng/mL. The analytes measured included benzoylecgonine (BZE), ecgonine methyl ester, cocaine, benzoylnorecgonine, norcocaine, m- and p-hydroxycocaine, and m- and p-hydroxybenzoylecgonine. Noncompartmental analysis was employed for the determination of plasma and saliva pharmacokinetic parameters. Urinary elimination half-lives for cocaine and metabolites were determined by constructing ARE (amount remaining to be excreted) plots. Two phases of urinary elimination of cocaine and metabolites were observed. An initial elimination phase was observed during withdrawal that was similar to the elimination pattern observed after acute dosing. The mean (N = 6) plasma, saliva, and urine cocaine elimination half-lives were 1.5 +/- 0.1 h, 1.2 +/- 0.2 h, and 4.1 +/- 0.9 h, respectively. For three subjects, the mean cocaine urinary elimination half-life for the terminal phase was 19.0 +/- 4.2 h. There was some difficulty in determining if a terminal elimination phase for cocaine was present for the remaining three subjects because of interference by high concentrations of BZE. A terminal elimination phase was also observed for cocaine metabolites with half-life estimates ranging from 14.6 to 52.4 h. These terminal elimination half-lives greatly exceeded previous estimates from studies of acute cocaine administration. These data suggest that cocaine accumulates in the body with chronic use resulting in a prolonged terminal elimination phase for cocaine and metabolites.     

Syntheses of metabolites of S-carboxymethyl-L-cysteine and S-methyl-L-cysteine and of some isotopically labelled (2H, 13C) analogues

The chemical syntheses of human metabolites of S-carboxymethyl-L-cysteine (3) and S-methyl-L-cysteine (12) are described. The additional preparation of some 2H- and 13C-labelled isotopomers enabled the direct evaluation of the stabilities of 3 and 12 under physiological conditions and also facilitated the unambiguous assignments of the signals in the 13C-NMR spectra of all compounds mentioned.     

Direct detection of antipyrine metabolites in rat urine by (13)C labeling and NMR spectroscopy.

Antipyrine is a useful probe to evaluate variation of in vivo activities of oxidative hepatic drug-metabolizing enzymes. Here we describe a new approach using (13)C labeling and NMR spectroscopy for the direct and simultaneous detection of all phase I and phase II metabolites of antipyrine in rat urine. [C-methyl-(13)C]Antipyrine was synthesized and administered orally to rats (100 mg/kg), and the 0- to 24-h postdose urine was analyzed by 100-MHz (13)C NMR spectroscopy under the conditions of distortionless enhancement by polarization transfer without any pretreatments such as deconjugation, chromatographic separation, and solvent extraction. Consequently, all the major metabolites in urine were successfully detected with favorable signal-to-noise ratios in the limited acquisition time (30 min). The assignments of the resonances were performed by enzymic modification and spiking authentic samples. The reproducibility of the NMR detection was sufficient for the quantitative evaluation of the metabolic profile. Effects of 3-methylcholanthrene on antipyrine metabolism were examined by this approach to evaluate variation of in vivo phase I and phase II metabolism of antipyrine in rats. The present approach is useful and practical to evaluate variation of in vivo activities of conjugation enzymes as well as oxidation enzymes responsible for the formation of antipyrine metabolites in rats. This direct approach would enhance the value of the antipyrine test because of the simplicity and convenience.     

Identification of the urinary metabolites of 4-bromoaniline and 4-bromo-[carbonyl-13C]-acetanilide in rat

1. The urinary excretion of 4-bromoaniline and its [carbonyl-(13)C]-labelled N-acetanilide, together with their corresponding metabolites, have been investigated in the rat following i.p. administration at 50 mg kg(-1). 2. Metabolite profiling was performed by reversed-phase HPLC with UV detection, whilst identification was performed using a combination of enzymic hydrolysis and directly coupled HPLC-NMR-MS analysis. The urinary metabolite profile was quantitatively and qualitatively similar for both compounds with little of either excreted unchanged. 3. The major metabolite present in urine was 2-amino-5-bromophenylsulphate, but, in addition, a number of metabolites with modification of the N-acetyl moiety were identified (from both the [(13)C]-acetanilide or produced following acetylation of the free bromoaniline). 4. For 4-bromoacetanilide, N-deacetylation was a major route of metabolism, but despite the detection of the acetanilide following the administration of the free aniline, there was no evidence of reacetylation (futile deacetylation). 5. Metabolites resulting from the oxidation of the acetyl group included a novel glucuronide of an N-glycolanilide, an unusual N-oxanilic acid and a novel N-acetyl cysteine conjugate.     

A physiological toxicokinetic model for exogenous and endogenous ethylene and ethylene oxide in rat, mouse, and human: formation of 2-hydroxyethyl adducts with hemoglobin and DNA

Ethylene (ET) is a gaseous olefin of considerable industrial importance. It is also ubiquitous in the environment and is produced in plants, mammals, and humans. Uptake of exogenous ET occurs via inhalation. ET is biotransformed to ethylene oxide (EO), which is also an important volatile industrial chemical. This epoxide forms hydroxyethyl adducts with macromolecules such as hemoglobin and DNA and is mutagenic in vivo and in vitro and carcinogenic in experimental animals. It is metabolically eliminated by epoxide hydrolase and glutathione S-transferase and a small fraction is exhaled unchanged. To estimate the body burden of EO in rodents and human resulting from exposures to EO and ET, we developed a physiological toxicokinetic model. It describes uptake of ET and EO following inhalation and intraperitoneal administration, endogenous production of ET, enzyme-mediated oxidation of ET to EO, bioavailability of EO, EO metabolism, and formation of 2-hydroxyethyl adducts of hemoglobin and DNA. The model includes compartments representing arterial, venous, and pulmonary blood, liver, muscle, fat, and richly perfused tissues. Partition coefficients and metabolic parameters were derived from experimental data or published values. Model simulations were compared with a series of data collected in rodents or humans. The model describes well the uptake, elimination, and endogenous production of ET in all three species. Simulations of EO concentrations in blood and exhaled air of rodents and humans exposed to EO or ET were in good agreement with measured data. Using published rate constants for the formation of 2-hydroxyethyl adducts with hemoglobin and DNA, adduct levels were predicted and compared with values reported. In humans, predicted hemoglobin adducts resulting from exposure to EO or ET are in agreement with measured values. In rodents, simulated and measured DNA adduct levels agreed generally well, but hemoglobin adducts were underpredicted by a factor of 2 to 3. Obviously, there are inconsistencies between measured DNA and hemoglobin adduct levels.     

Association of CYP2E1, GST and mEH genetic polymorphisms with urinary acrylamide metabolites in workers exposed to acrylamide

This study elucidates the association of acrylamide metabolites, N-acetyl-S-(2-carbamoylethyl)-cysteine (AAMA), N-acetyl-S-(1-carbamoyl-2-hydroxyethyl)-cysteine (GAMA2), and N-acetyl-S-(2-carbamoyl-2-hydroxyethyl)-cysteine (GAMA3) in urine with genetic polymorphisms of the metabolic enzymes cytochrome P450 2E1 (CYP2E1), microsomal epoxide hydrolase (mEH) in exon 3 and exon 4, glutathione transferase theta (GSTT1) and mu (GSTM1), involved in the activation and detoxification of acrylamide (AA) in humans. Eighty-five workers were recruited, including 51 AA-exposed workers and 34 administrative staffs serve as controls. Personal air sampling was performed for the exposed workers. Each subject provided pre- and post-shift urine samples and blood samples. Urinary AAMA, GAMA2 and GAMA3 levels were simultaneously quantified using liquid chromatography-electronspray ionization/tandem mass spectrometry (LC-ESI-MS/MS). CYP2E1, mEH (in exon 3 and exon 4), GSTT1, and GSTM1 were analyzed using polymerase chain reaction (PCR). Our results reveal that AA personal exposures ranged from 4.37 × 10⁻³ to 113.61 μg/m³ with a mean at 15.36 μg/m³. The AAMA, GAMA2, and GAMA3 levels in the exposed group significantly exceeded those in controls. The GAMAs (the sum of GAMA2 and GAMA3)/AAMA ratios, potentially reflecting the proportion of AA metabolized to glycidamide (GA), varied from 0.003 to 0.456, and indicate high inter-individual variability in the metabolism of AA to GA in this study population. Multivariate regression analysis demonstrates that GSTM1 genotypes significantly modify the excretion of urinary AAMA and the GAMAs/AAMA ratio, exon 4 of mEH was significantly associated with the urinary GAMAs levels after adjustment for AA exposures. These results suggest that mEH and/or GSTM1 may be associated with the formation of urinary AAMA and GAMAs. Further study may be needed to shed light on the role of both enzymes in AA metabolism.