Fluoroacetylation/fluoroethylesterification as a derivatization approach for gas chromatography-mass spectrometry in metabolomics: preliminary study of lymphohyperplastic diseases.

Metabolic fingerprinting in combination with gas chromatography and multivariate analysis is being extensively employed for the improved understanding of biological changes induced by endogenous or exogenous factors. Chemical derivatization increases the sensitivity and specificity of gas chromatography-mass spectrometry (GC-MS) for polar or thermally labile biological compounds, which bear derivatizable groups. Thus, there is a constant demand for simple methods of derivatization and separation that satisfy the need for metabolite analysis, identifying as many chemical classes of compounds as possible. In this study, an optimized protocol of extraction and derivatization is established as a generally applicable method for the analysis of a wide range of classes of metabolites in urine, whole blood and saliva. Compounds of biological relevance bearing hydroxyl- carboxyl- and amino-groups are derivatized using single-step fluoroacetylation/fluoroethylesterification after proper optimization of the protocol. Subsequently, the developed derivatization procedure is engaged in finding blood metabolic biomarkers, induced by lymphohyperplastic disease, through the metabolomic fingerprinting approach, the multivariate modeling (hierarchical cluster analysis) and GC-MS. Our preliminary, GC-MS-based metabolomic fingerprinting study underlines the contribution of certain metabolites to the discrimination of patients with lymphohyperplastic diseases.

Radiation dosimetry and biodistribution of the hypoxia tracer (18)F-EF5 in oncologic patients.

UNLABELLED: The primary goals of this study were to determine the biodistribution and excretion of (18)F-EF5 in oncologic patients, to estimate the radiation-absorbed dose and to determine the safety of this drug. METHODS: Sixteen patients with histologically confirmed malignancy received a mean intravenous infusion of 217 MBq (range 107-364 MBq) of (18)F-EF5. Over a 4-6-hour period, four to five serial positron emission tomography (PET) or PET/computed tomography (CT) scans were obtained. To calculate the radiation dosimetry estimates, volumes of interest were drawn over the source organs for each PET scan or on the CT for each PET/CT scan. Serial blood samples were obtained to measure (18)F-EF5 blood clearance. Bladder-wall dose was calculated based on urine activity measurements. RESULTS: The urinary bladder received the largest radiation-absorbed dose, 0.12 ± 0.034 mSv/MBq (mean ± SD). The average effective dose equivalent and the effective dose of (18)F-EF5 were 0.021 ± 0.003 mSv/MBq and 0.018 ± 0.002 mSv/MBq, respectively. (18)F-EF5 was well tolerated in all subjects. CONCLUSIONS: (18)F-EF5 was demonstrated to be safe for patients, and the radiation exposure is clinically acceptable. As with any radiotracer with primary excretion in the urine, the bladder-wall dose can be minimized by active hydration and frequent voiding.

Use of the Rey-Osterrieth Complex Figure Test for neurotoxicity evaluation of mixtures in children.

The aim of this study was to assess the value of the children's version of the Rey-Osterrieth Complex Figure Test as a screening test in a population exposed to different mixtures of neurotoxicants. Copy and Immediate Recall scores were evaluated through the test. Children were recruited from three sites; an area with natural contamination by fluoride and arsenic (F-As), a mining-metallurgical area with lead and arsenic contamination (Pb-As) and a malaria zone with the evidence of fish contaminated with dichlorodiphenyltrichloroethane (DDT) and polychlorinated biphenyls (PCBs). Children aged 6-11 years old, living in one of the three polluted sites since birth were recruited (n=166). The exposure was evaluated as follows: fluoride and arsenic in urine, lead in blood and DDT, dichlorodiphenyldichloroethylene (DDE) and PCBs in serum. To evaluate the test performance, z-scores for Copy and Immediate Recall were calculated. The proportion of children by residence area with performance lower than expected by age (below -1 SD) for Copy and Immediate Recall was in the F-As area (88.7% and 59%) and in the DDT-PCBs area (73% and 43.8%), respectively. In the Pb-As area, the proportion was 62% for both tests. After adjustment, Copy correlated inversely with fluoride in urine (r=-0.29; p<0.001) and Immediate Recall correlated inversely with fluoride in urine (r=-0.27; p<0.05), lead in blood (r=-0.72; p<0.01), arsenic in urine (r=-0.63; p<0.05) and DDE (r=-0.25; p<0.05). This study provided evidence that children included in this research are living in high risk areas and were exposed to neurotoxicants. Poor performance in the test could be explained in some way by F, Pb, As or DDE exposure, however social factors or the low quality of school education prevalent in the areas could be playing an important role.

Disposition of perfluorinated acid isomers in Sprague-Dawley rats; part 1: single dose.

Perfluorinated acids (PFAs) and their precursors (PFA-precursors) exist in the environment as linear and multiple branched isomers. These isomers are hypothesized to have different biological properties, but no isomer-specific data are currently available. The present study is the first in a two-part project examining PFA isomer-specific uptake, tissue distribution, and elimination in a rodent model. Seven male Sprague-Dawley rats were administered a single gavage dose of approximately 500 microg/kg body weight perfluorooctane sulfonate (C(8)F(17)SO(3)(-), PFOS), perfluorooctanoic acid (C(7)F(15)CO(2)H, PFOA), and perfluorononanoic acid (C(8)F(17)CO(2)H, PFNA) and 30 microg/kg body weight perfluorohexane sulfonate (C(6)F(13)SO(3)(-), PFHxS). Over the subsequent 38 d, urine, feces, and tail-vein blood samples were collected intermittently, while larger blood volumes and tissues were collected on days 3 and 38 for isomer analysis by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). For all PFAs, branched isomers generally had lower blood depuration half-lives than the corresponding linear isomer. The most remarkable exception was for the PFOS isomer containing an alpha-perfluoromethyl branch (1m-PFOS), which was threefold more persistent than linear PFOS, possibly due to steric shielding of the hydrophilic sulfonate moiety. For perfluoromonomethyl-branched isomers of PFOS, a structure-property relationship was observed whereby branching toward the sulfonate end of the perfluoroalkyl chain resulted in increased half-lives. For PFHxS, PFOA, and PFOS, preferential elimination of branched isomers occurred primarily via urine, whereas for PFNA preferential elimination of the isopropyl isomer occurred via both urine and feces. Changes in the blood isomer profiles over time and their inverse correlation to isomer elimination patterns in urine, feces, or both provided unequivocal evidence of significant isomer-specific biological handling. Source assignment based on PFA isomer profiles in biota must therefore be conducted with caution, because isomer profiles are unlikely to be conserved in biological samples.

Disposition of perfluorinated acid isomers in Sprague-Dawley rats; part 2: subchronic dose.

Two major industrial synthetic pathways have been used to produce perfluorinated acids (PFAs) or their precursors: Telomerization and electrochemical fluorination (ECF). Products of telomer and ECF origin can be distinguished by structural isomer profiles. A mixture of linear and branched perfluoroalkyl isomers is associated with ECF. Telomer products characteristically consist of a single perfluoroalkyl geometry, typically linear. In biota, it is unclear if the isomer profile is conserved relative to the exposure medium and hence whether PFA isomer profiles in organisms are useful for distinguishing environmental PFA sources. A companion study suggested isomer-specific disposition following a single oral gavage exposure to rats. To confirm these findings under a more realistic subchronic feeding scenario, male and female rats were administered PFA isomers by diet for 12 weeks, followed by a 12-week depuration period. The diet contained 500 ng/g each of ECF perfluorooctanoate (PFOA, approximately 80% n-PFOA), ECF perfluorooctane sulfonate (PFOS, approximately 70% n-PFOS), and linear and isopropyl perfluorononanoate (n- and iso-PFNA). Blood sampling during the exposure phase revealed preferential accumulation of n-PFOA and n-PFNA compared to most branched isomers. Female rats depurated all isomers faster than males. Both sexes eliminated most branched perfluorocarboxylate isomers more rapidly than the n-isomer. Elimination rates of the major branched PFOS isomers were not statistically different from n-PFOS. Two minor isomers of ECF PFOA and one branched PFOS isomer had longer elimination half-lives than the n-isomers. Although extrapolation of these pharmacokinetics trends in rats to humans and wildlife requires careful consideration of dosage level and species-specific physiology, cumulative evidence suggests that perfluorocarboxylate isomer profiles in biota may not be suitable for quantifying the relative contributions of telomer and ECF sources.

The pharmacokinetics of JS-38, a novel antineoplastic drug, in rats.

To evaluate the pre-clinical pharmacokinetics of JS-38(C22H1404N2S2F6, MW: 548), a study was conducted in Wistar rats (3 female, 2 male: 200-250 g about 6 or 7 months). The concentration-time curve of JS-38 in rats demonstrated the pharmacokinetic (PK) characteristics of a two-compartmental model. The area under the concentration-time curve from zero to infinity (AUC(0-infinity)) for the low, middle and high dosage (i.e., 20, 50 and 125 mg x kg(-1)) amounted to 46.850 +/- 19.946, 161.101 +/- 58.877 and 312.565 +/- 187.273 mg/L x h respectively; a positive correlation was demonstrated between the AUC(0-infinity). and the dosages in question (r = 0.99). The average time to reach maximum concentration (Tmax) was 3.( RSD: 20.4% and the half-life (t(1/2)) was 11.4 h( RSD: 8.8% P > 0.05. For the low, middle and high dosage, the maximum concentration (Cmax) was 4.882, 11.248, and 13.431 microg x mL(-1) respectively. After the administration of JS-38, except for the brain and spinal marrow, the drug distribution in the different body tissues varied, in particular in the liver, intestine and thyroid gland. A significant distribution of JS-38 was detected in cancerous tissues, and its concentrations demonstrated a tendency increase over time. There was a certain degree of distribution in the bone marrow. The urine samples showed that JS-38 nearly was practically not eliminated in its original form. The amount eliminated after 72h via the bile was only 1.03 +/- 0.1% of the administered dose. In the rat model, most of the JS-38 in its original form (53.58 +/- 22.28%) was excreted via the feces. When the intragastric administration of doses of 20, 50 and 125 mg x kg(-1) was compared with i.v. administered JS-38 (1 mg x kg(-1)), the absolute bioavailability amounted to 22.2 +/- 9.5%, 30.4 +/- 14.5% and 23.6 +/- 11.3% respectively. It was found that this compound is well absorbed in to the system and that it shows favorable PK properties. The outcome of this early pre-clinical study indicates that JS-38 is a promising drug candidate for further development.

Experimental exposure to 1,1,1-trifluoroethane (HFC-143a): uptake, disposition and acute effects in male volunteers.

The aim of this study was to determine the toxicokinetics and some effects of 1,1,1-trifluoroethane (HFC-143a) in humans. Nine male volunteers were experimentally exposed to 500ppm HFC-143a for 2h during light physical exercise (50W) in an exposure chamber. Blood, urine and exhaled air were sampled before, during and up to 19h after exposure and analysed for HFC-143a by gas chromatography. These data were described by a physiologically based toxicokinetic (PBTK) model. The electrocardiograms of the volunteers were monitored during exposure. Before, during and after exposure the volunteers rated symptoms related to irritation and CNS-symptoms on a visual analogue scale. Inflammatory markers (C-reactive protein, serum amyloid A protein, D-dimer, fibrinogen) and uric acid were analysed in plasma collected before and 21h after exposure. The exposures were performed after informed consent and ethical approval. The plasma concentration of HFC-143a increased promptly at start of exposure, and decreased in the same manner post-exposure. A stable level of 4.8+/-2.0 microM (mean+/-S.D.) was reached within 30min of exposure. The HFC-143a concentration in plasma and exhaled air decreased fast and in parallel when exposure was stopped. The urinary excretion of HFC-143a after exposure was 0.0007% of the inhaled amount. The half-time in urine, calculated from pooled data, was 53min. The experimental and simulated time courses in blood and exhaled air were in agreement. The simulated relative uptake during the exposure was 1.6+/-0.3%. The fibrinogen level in plasma had increased by 11% 1 day post-exposure. No statistically significant increase was seen for the other inflammatory markers or for uric acid. No effects of exposure were seen either in the electrocardiographic monitorings or as symptom ratings on the visual analogue scale.

Influence of sevoflurane on the metabolism and renal effects of compound A in rats.

BACKGROUND: The sevoflurane degradation product compound A is nephrotoxic in rats. In contrast, patient exposure to compound A during sevoflurane anesthesia has no clinically significant renal effects. The mechanism for this difference is incompletely understood. One possibility is that the metabolism and toxicity of compound A in humans is prevented by sevoflurane. However, the effect of sevoflurane on compound A metabolism and nephrotoxicity is unknown. Thus, the purpose of this investigation was to determine the effect of sevoflurane on the metabolism and renal toxicity of compound A in rats. METHODS: Male rats received 0.25 mmol/kg intraperitoneal compound A, alone and during sevoflurane anesthesia (3%, 1.3 minimum alveolar concentration, for 3 h). Compound A metabolites in urine were quantified, and renal function was evaluated by serum creatinine and urea nitrogen, urine volume, osmolality, protein excretion, and renal tubular histology. RESULTS: Sevoflurane coadministration with compound A inhibited compound A defluorination while increasing relative metabolism through pathways of sulfoxidation and beta-lyase-catalyzed metabolism, which mediate toxicity. Sevoflurane coadministration with compound A increased some (serum creatinine and urea nitrogen, and necrosis) but not other (urine volume, osmolality, and protein excretion) indices of renal toxicity. CONCLUSIONS: Sevoflurane does not suppress compound A nephrotoxicity in rats in vivo. These results do not suggest that lack of nephrotoxicity in surgical patients exposed to compound A during sevoflurane anesthesia results from an inhibitory effect of sevoflurane on compound A metabolism and toxicity. Rather, these results are consistent with differences between rats and humans in compound A exposure and inherent susceptibility to compound A nephrotoxicity.

Comparison of the elimination between perfluorinated fatty acids with different carbon chain length in rats.

Elimination in urine and feces was compared between four perfluorinated fatty acids (PFCAs) with different carbon chain length. In male rats, perfluoroheptanoic acid (PFHA) was rapidly eliminated in urine with the proportion of 92% of the dose being eliminated within 120 h after an intraperitoneal injection. Perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA) and perfluorodecanoic acid (PFDA) was eliminated in urine with the proportions of 55, 2.0 and 0.2% of the dose, respectively. By contrast, four PFCAs were eliminated in feces with the proportion of less than 5% of the dose within 120 h after an injection. In female rats, the proportions of PFOA and PFNA eliminated in urine within 120 h were 80% and 51% of the dose, respectively, which were significantly higher compared with those in male rats. There was the tendency that PFCA with longer carbon chain length is less eliminated in urine in both male and female rats. Fecal elimination of PFCAs was not different between PFCAs in female rats and comparable to those in male rats. The rates of biliary excretion of PFCAs in male rats were slower than those in female rats. Sex-related difference in urinary elimination of PFOA was abolished when male rats had been castrated. On the contrary, treatment with testosterone suppressed the elimination of PFOA in urine in both castrated male rats and female rats. The effect of testosterone was in a time- and dose-dependent manner. These results suggest that PFCAs are distinguished by their carbon chain length by a renal excretion system, which is regulated by testosterone.

Compound A uptake and metabolism to mercapturic acids and 3,3,3-trifluoro-2-fluoromethoxypropanoic acid during low-flow sevoflurane anesthesia: biomarkers for exposure, risk assessment, and interspecies comparison.

BACKGROUND: Sevoflurane is degraded during low-flow anesthesia to fluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinyl ether ("compound A"), which causes renal necrosis in rats but is not known to cause nephrotoxicity in surgical patients. Compound A is metabolized to glutathione S-conjugates and then to cysteine S-conjugates, which are N-acetylated to mercapturic acids (detoxication pathway), or metabolized by renal beta-lyase to reactive intermediates (toxification pathway) and excreted as 3,3,3-trifluoro-2-fluoromethoxypropanoic acid. This investigation quantified compound A metabolites in urine after low-flow sevoflurane administration, to assess relative flux via these two pathways. METHODS: Patients (n = 21) with normal renal function underwent low-flow (11 min) sevoflurane anesthesia designed to maximize compound A formation. Inspiratory, expiratory, and alveolar compound A concentrations were quantified. Urine mercapturic acids and 3,3,3-trifluoro-2-fluoromethoxypropanoic acid concentrations were measured by gas chromatography and mass spectrometry. RESULTS: Sevoflurane exposure was 3.7 +/- 2.0 MAC-h. Inspired compound A maximum was 29 +/- 14 ppm; area under the inspired concentration versus time curve (AUCinsp) was 78 +/- 58 ppm x h. Compound A dose, calculated from pulmonary uptake, was 0.39 +/- 0.35 mmol (4.8 +/- 4.0 micromol/kg) and correlated with AUCinsp (r2 = 0.84, P < 0.001). Mercapturic acids excretion was complete after 2 days, whereas 3,3,3-trifluoro-2-fluoromethoxypropanoic acid excretion continued for 3 days in some patients. Total (3-day) mercapturates and fluoromethoxypropanoic acid excretion was 95 +/- 49 and 294 +/- 416 micromol, respectively (1.2 +/- 0.6 and 3.6 +/- 5.0 micromol/kg). CONCLUSION: Compound A doses during 3.7 MAC-h, low-flow sevoflurane administration in humans are substantially less than the threshold for renal toxicity in rats (200 micromol/kg). Compound A metabolites quantification may provide a biomarker for compound A exposure and relative metabolism via toxification and detoxication pathways. Compared with previous investigations, relative metabolic flux (fluoromethoxypropanoic acid/mercapturates) through the toxification pathway was sixfold greater in rats than in humans. Species differences in dose and metabolism may influence compound A renal effects.

Cysteine conjugate beta-lyase-dependent metabolism of compound A (2-[fluoromethoxy]-1,1,3,3,3-pentafluoro-1-propene) in human subjects anesthetized with sevoflurane and in rats given compound A.

BACKGROUND: Sevoflurane undergoes Baralyme- or soda lime-catalyzed degradation in the anesthesia circuit to yield compound A (2-[fluoromethoxy]-1,1,3,3,3-pentafluroro-1-propene), which is nephrotoxic in rats and undergoes metabolism via the cysteine conjugate beta-lyase pathway in those animals. The objective of these experiments was to test the hypothesis that compound A undergoes beta-lyase-dependent metabolism in humans. METHODS: Human volunteers were anesthetized with sevoflurane (1.25 minimum alveolar concentration, 3%, 2 l/min, 8 h) and thereby exposed to compound A. Urine was collected at 24-h intervals for 72 h after anesthesia. Rats, which served as a positive control, were given compound A intraperitoneally, and urine was collected for 24 h afterward. Human and rat urine samples were analyzed by 19F nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry for the presence of compound A metabolites. RESULTS: Analysis of human and rat urine showed the presence of the compound A metabolites S-[2(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-N-acetyl-L- cysteine, (E)- and (Z)-S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl- L-cysteine, 2-(fluoromethoxy)-3,3,3-trifluoropropanoic acid, 3,3,3-trifluorolactic acid, and inorganic fluoride. The presence of 2-(fluoromethoxy)3,3,3-trifluoropropanoic acid and 3,3,3-trifluorolactic acid in human urine was confirmed by gas chromatography-mass spectrometry. CONCLUSIONS: The formation of compound A-derived mercapturates shows that compound A undergoes glutathione S-conjugate formation. The identification of 2-(fluoromethoxy)-3,3,3-trifluoropropanoic acid and 3,3,3-trifluorolactic acid in the urine of humans anesthetized with sevoflurane shows that compound A undergoes beta-lyase-dependent metabolism. Metabolite formation was qualitatively similar in both human volunteers anesthetized with sevoflurane, and thereby exposed to compound A, and in rats given compound A, indicating that compound A is metabolized by the beta-lyase pathway in both species.

Disposition in rat of a new fluorinated, biocompatible, non-ionic telomeric carrier.

1. The disposition of the new fluorinated, biocompatible, non-ionic telomeric carrier trisacryl conjugate (F-TAC) labelled with 13C and 14C on the amide function has been studied in the rat after p.o. and i.v. administration. 2. After i.v. administration, excretion measurements have shown that radioactivity was eliminated mainly in the urine (69% within 24 h), and that faecal excretion was low (8% within 72 h). After p.o. administration, faecal elimination was significantly increased (30% within 72 h). No radioactivity was eliminated as 14CO2, after either route of administration. 3. After i.v. administration, plasma radioactivity exhibited a biphasic decrease, with t1/2 = 20 min for the first phase and 29.5 h for the second phase. The maximal plasma concentration was obtained 40 min after oral dosing, followed by a monoexponential decrease with t1/2 = 38.1 h. The ratio AUC (p.o.)/AUC (i.v.) as an assessment of bioavailability was 0.22. 4. After both i.v. and p.o. administration, a relatively homogeneous concentration of radioactivity was found in most organs, close or below the plasma concentration, indicating that tissues do not exhibit a high affinity for this molecule. In addition, F-TAC did not cross the blood-brain barrier. 5. Analysis of urine and plasma on DOWEX AG1X2 anionic resin showed that < 20% of the radioactivity was bound to this support. 13C- and 19F-nmr analysis of the non-bound radioactivity identified it to unchanged F-TAC, with bound radioactivity being due to polyanionic telomers arising from the hydrolysis of the amide function.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolic fate and disposition of 1,1,1,2-tetrafluoroethane (HFC134a) in rat following a single exposure by inhalation.

1. The metabolic fate and disposition of [U-14C]-1,1,1,2-tetrafluoroethane ([U-14C]-HFC134a) has been determined in the male and female rat following a 1 h single exposure by inhalation to atmospheres of 10,000 ppm. 2. Of the inhaled dose, approx. 1% was recovered in urine, faeces and expired air postexposure indicating that absorption of this fluorocarbon across the lung is poor. Of this 1%, approx, two-thirds were exhaled within 1 h of the cessation of exposure as unchanged HFC134a. The remaining radioactivity was exhaled as [14C]-carbon dioxide or excreted in urine and faeces as trifluoroacetic acid. 3. Carbon dioxide was the major metabolite of HFC134a accounting for 0.22 and 0.27% of the inhaled dose in the male and female rat, respectively. Urinary excretion accounted for 0.09% of the dose and faecal excretion 0.04% of the dose by both sexes. 4. Total metabolism measured as the sum of the radioactivities in urine, faeces and as carbon dioxide amounted to 0.34 and 0.40% of the inhaled dose in male and female, respectively. 5. There were no major sex differences in the rates, routes or amounts of radiolabel excreted. Analysis of a range of tissues at 5 days postexposure showed a relatively uniform distribution of radioactivity. There was no evidence for a specific uptake of HFC134a or a metabolite into any organ or tissue analysed, including fat.

Metabolism of L-cysteine S-conjugates and N-(trideuteroacetyl)-L-cysteine S-conjugates of four fluoroethylenes in the rat. Role of balance of deacetylation and acetylation in relation to the nephrotoxicity of mercapturic acids.

The relationship between the relative nephrotoxicity of the mercapturic acids (NAc) of the fluorinated ethylenes tetrafluoroethylene (TFE), chlorotrifluoroethylene (CTFE), 1,1-dichloro-2,2-difluoroethylene (DCDFE) and 1,1-dibromo-2,2-difluoroethylene (DBDFE), and the biotransformation by activating (N-deacetylase and beta-lyase) and inactivating (N-acetyltransferase) enzymes was studied in the rat. After intraperitoneal (i.p.) administration of 50 mumol/kg of N-(trideuteroacetyl)-labeled mercapturic acids of DCDFE and DBDFE to rats, significant amounts of the dose were excreted unchanged: with DCDFE-NAc, 17% of the dose, and DBDFE-NAc, 31% of the dose. In contrast, the corresponding deuterium-labeled mercapturic acids of TFE and CTFE were excreted unchanged at less than 1% of the dose. With DCDFE-NAc and DBDFE-NAc, also high amounts of unlabeled mercapturic acids were excreted, respectively 48% and 28% of the dose, indicating extensive N-deacetylation followed by reacetylation in vivo. Only small amounts (less than 2%) of unlabeled mercapturic acids were excreted with TFE-NAc and CTFE-NAc. After administration of the cysteine S-conjugates DCDFE-Cys and DBDFE-Cys to rats, high amounts of the corresponding mercapturic acids were detected in urine, respectively 57% and 45% of the dose. After administration of TFE-Cys and CTFE-Cys, however, only small amounts were excreted as the corresponding mercapturic acid, approximately 4% of the dose. The strongly different amounts of mercapturic acids in urine may be attributed to the strong differences in N-deacetylation activities which were found in rat renal fractions. The threshold dose of the mercapturic acids to cause nephrotoxicity in male Wistar rats increased in the order: CTFE-NAc (25 mumol/kg) less than TFE-NAc (50 mumol/kg) less than DCDFE-NAc (75 mumol/kg) less than DBDFE-NAc (100 mumol/kg). A higher ratio of N-deacetylation and N-acetylation activities, resulting in a higher availability of cysteine S-conjugate, in addition to a higher specific activity of cysteine S-conjugate beta-lyase, probably explains the higher nephrotoxicity of TFE-NAc and CTFE-NAc when compared to DCDFE-NAc and DBDFE-NAc. The much lower activities of N-deacetylation and beta-lyase which are observed in hepatic fractions may explain the lack of hepatotoxicity of the mercapturic acids studied.

Nephrotoxicity and hepatotoxicity of 1,1-dichloro-2,2-difluoroethylene in the rat. Indications for differential mechanisms of bioactivation.

1,1-Dichloro-2,2-difluoroethylene (DCDFE) produced marked nephrotoxicity in rats upon an i.p. dose of 150 mumole/kg. At doses higher than 375 mumole/kg, DCDFE also produced hepatotoxicity. Aminooxyacetic acid, an inhibitor of cysteine conjugate beta-lyase, appeared to be slightly nephrotoxic in Wistar rats. Nevertheless it exerted an inhibitory effect on the nephrotoxicity of DCDFE. The N-acetylcysteine conjugate of DCDFE was identified as a major urinary metabolite of DCDFE. When administered as such, this conjugate appeared to be a potent nephrotoxin, without any effect on the liver, indicating that glutathione conjugation of DCDFE is most likely a bioactivation step for nephrotoxicity. The appearance of traces of chlorodifluoroacetic acid in urine of rats treated with higher doses of DCDFE indicates the existence of an oxidative pathway of metabolism of DCDFE, probably involving epoxidation by hepatic mixed-function oxidases. It is speculated that the latter route might account for the hepatotoxicity at higher doses of DCDFE. The nephro- and hepatotoxicity of DCDFE, therefore, most likely are the result of two different mechanisms of bioactivation.

Mass spectral identification of the metabolites of alpha,alpha-dimethyl-4-(alpha,alpha,beta,beta-tetrafluorophenethyl)-benzylamine (MK-251), a novel antiarrhythmic agent, in various species.

The identification of a number of metabolites of the novel antiarrhythmic agent, alpha,alpha-dimethyl-4-(alpha,alpha,beta,beta-tetrafluorophenethyl)benzylamine (MK-251), is presented. The compound is extensively metabolized by dog, monkey, baboon and man. Similar metabolic profiles were obtained for all species. Isolation and purification were accomplished by solvent extraction and chromatographic (column, gas and thin-layer) procedures. Gas chromatography, derivatization, infrared, nuclear magnetic resonance and particularly combined gas chromatography low and high resolution mass spectrometry techniques were employed to characterize the metabolites. The major urinary and plasma metabolites were identified as 2-[4-(alpha,alpha,beta,beta-tetrafluorophenethyl)phenyl]-2-propanol and its glucuronide conjugate. Other metabolites characterized were: the N-glucuronide of MK-251; 2-[4-(alpha,alpha,beta,beta-tetrafluorophenethyl)phenyl]propene; 2-nitro-2-[4-)alpha,alpha,beta,beta-tetrafluorophenethyl)phenyl]propane; alpha,alpha-dimethyl-4(alpha,alpha,beta,beta-tetrafluorophenethyl)benzyl methyl ether; and 4-(alpha,alpha,beta,beta-tetrafluorophenethyl)acetophenone. The 0-methyl ether metabolite represents the first instance of in vivo alkylation of a tertiary alcohol. Tentative identification was made for the N-hydroxy analog of MK-251 and for the glycol analog of 2-[4-(alpha,alpha,beta,beta-tetrafluorophenethyl)phenyl]-2-propanol. The observed pharmacological response appears to result mainly from MK-251 and not from the four metabolites.

Physiological disposition and metabolic fate of a new antiarrhythmic agent, alpha, alpha-dimethyl-4-(alpha, alpha, beta, beta-tetrafluorophenethyl) benzylamine in the rat, dog, monkey, baboon, and man.

The physiological disposition of a new orally active antiarrhythmic drug, alpha, alpha-dimethyl-4-(alpha, alpha, beta, beta-tetrafluorophenethyl)benzylamine (MK-251) was investigated in the rat, dog, rhesus monkey, baboon, and man. MK-251 was extensively absorbed after oral administration in all species. Fecal excretion was the major route of tracer elimination in the rat (70%) and dog (80%), whereas the monkey, baboon, and man excreted the majority of the dose via the urine (40-80%). MK-251 and/or its metabolites were widely distributed in rat tissues and exhibited tissue/plasma ratios greater than one in most instances. The lung, liver, and kidney possessed a high tissue affinity for drug and metabolites. The plasma and urinary profile of radioactivity indicated extensive metabolism of MK-251 in all species. Less than 5% of the plasma and urinary radioactivity was identified as unchanged drug. In spite of extensive metabolic transformations, a remarkable feature of this drug is its persistence in the plasma for long periods of time. This is thought to be due to tissue affinity. The metabolic pattern for MK-251 was essentially the same in all species. The major metabolites present in the plasma and the urine were identified as the carbinol analog of MK-251, 2-[4-(alpha, alpha, beta, beta-tetrafluorophenethyl)phenyl]-2-propanol (I), and its glucuronide conjugate. Other metabolites characterized in the urine and plasma were: the N-glucuronide of MK-251, 2-[4-(alpha, alpha, beta, beta-tetrafluorophenethyl)phenyl]propene (II), 2-nitro-2-[4-(alpha, alpha, beta, beta-tetrafluorophenethyl)phenyl]propane (III), alpha, alpha-dimethyl-4-(alpha, alpha, beta, beta-tetrafluorophenethyl)benzyl methyl ether (IV-1) and 4-(alpha, alpha, beta, beta-tetrafluorophenethyl), acetophenone (IV-2). Two minor urinary metabolites were tentatively identified as the N-hydroxy analog of MK-251 and the glycol analog of carbinol I. The in vivo formation of the methyl ether represents the first report of alkylation of a tertiary alcohol.