Metabolism of [14C]methamphetamine in man, the guinea pig and the rat

1. The metabolites of (+/-)-2-methylamino-1-phenyl[1-(14)C]propane ([(14)C]methamphetamine) in urine were examined in man, rat and guinea pig. 2. In two male human subjects receiving the drug orally (20mg per person) about 90% of the (14)C was excreted in the urine in 4 days. The urine of the first day was examined for metabolites, and the main metabolites were the unchanged drug (22% of the dose) and 4-hydroxymethamphetamine (15%). Minor metabolites were hippuric acid, norephedrine, 4-hydroxyamphetamine, 4-hydroxynorephedrine and an acid-labile precursor of benzyl methyl ketone. 3. In the rat some 82% of the dose of (14)C (45mg/kg) was excreted in the urine and 2-3% in the faeces in 3-4 days. In 2 days the main metabolites in the urine were 4-hydroxymethamphetamine (31% of dose), 4-hydroxynorephedrine (16%) and unchanged drug (11%). Minor metabolites were amphetamine, 4-hydroxyamphetamine and benzoic acid. 4. The guinea pig was injected intraperitoneally with the drug at two doses, 10 and 45mg/kg. In both cases nearly 90% of the (14)C was excreted, mainly in the urine after the lower dose, but in the urine (69%) and faeces (18%) after the higher dose. The main metabolites in the guinea pig were benzoic acid and its conjugates. Minor metabolites were unchanged drug, amphetamine, norephedrine, an acid-labile precursor of benzyl methyl ketone and an unknown weakly acidic metabolite. The output of norephedrine was dose-dependent, being about 19% on the higher dose and about 1% on the lower dose. 5. Marked species differences in the metabolism of methamphetamine were observed. The main reaction in the rat was aromatic hydroxylation, in the guinea pig demethylation and deamination, whereas in man much of the drug, possibly one-half, was excreted unchanged.     

Complete reductive dechlorination and mineralization of pentachlorophenol by anaerobic microorganisms.

Anaerobically digested municipal sewage sludge which had been acclimated to monochlorophenol degradation for more than 2 years was shown to degrade pentachlorophenol (PCP). Di-, tri-, and tetrachlorophenols accumulated when PCP was added to the individual acclimated sludges. When the 2-chlorophenol- (2-CP), 3-CP-, and 4-CP-acclimated sludges were mixed in equal volumes, PCP was completely dechlorinated. The same results were obtained in sludge acclimated to the three monochlorophenol isomers simultaneously. With repeated PCP additions, 3,4,5,-trichlorophenol, 3,5-dichlorophenol, and 3-CP accumulated in less than stoichiometric amounts. All chlorinated compounds disappeared after PCP additions were stopped. All chlorinated compounds disappeared after PCP additions were stopped. Incubations with [14C]PCP resulted in 66% of the added 14C being mineralized to 14CO2 and 14CH4. Technical-grade PCP was found to be degraded initially at a rate very similar to that of reagent-grade PCP, but after repeated additions, the technical PCP was degraded more slowly. Pentabromophenol was also rapidly degraded by the mixture of acclimated sludges. These results clearly show the complete reductive dechlorination of PCP by the combined activities of three chlorophenol-degrading populations.     

Complete reductive dechlorination and mineralization of pentachlorophenol by anaerobic microorganisms

Anaerobically digested municipal sewage sludge which had been acclimated to monochlorophenol degradation for more than 2 years was shown to degrade pentachlorophenol (PCP). Di-, tri-, and tetrachlorophenols accumulated when PCP was added to the individual acclimated sludges. When the 2-chlorophenol- (2-CP), 3-CP-, and 4-CP-acclimated sludges were mixed in equal volumes, PCP was completely dechlorinated. The same results were obtained in sludge acclimated to the three monochlorophenol isomers simultaneously. With repeated PCP additions, 3,4,5,-trichlorophenol, 3,5-dichlorophenol, and 3-CP accumulated in less than stoichiometric amounts. All chlorinated compounds disappeared after PCP additions were stopped. All chlorinated compounds disappeared after PCP additions were stopped. Incubations with [14C]PCP resulted in 66% of the added 14C being mineralized to 14CO2 and 14CH4. Technical-grade PCP was found to be degraded initially at a rate very similar to that of reagent-grade PCP, but after repeated additions, the technical PCP was degraded more slowly. Pentabromophenol was also rapidly degraded by the mixture of acclimated sludges. These results clearly show the complete reductive dechlorination of PCP by the combined activities of three chlorophenol-degrading populations.     

Species differences in the metabolism of norephedrine in man, rabbit and rat

1. (+/-)-2-Amino-1-phenyl[1-(14)C]propan-1-ol ([(14)C]norephedrine) was administered orally to man, rat and rabbit and the metabolites excreted in the urine were identified and measured. Pronounced species differences in the metabolism of the drug were found. 2. Three male human subjects, receiving 25mg each of [(14)C]norephedrine hydrochloride, excreted over 90% of the (14)C in the first day. The main metabolite was the unchanged drug (86% of the dose) and minor metabolites were hippuric acid and 4-hydroxynorephedrine. 3. In rats given 12mg of the drug/kg almost 80% of the (14)C administered was excreted in the first day. The major metabolites in the urine were the unchanged drug (48% of the dose), 4-hydroxynorephedrine (28%) and trace amounts of side-chain degradation products. 4. Rabbits given 12mg of the drug/kg excreted 85-95% of the dose of (14)C in the urine in the first 24h after dosing. The major metabolites in the urine were conjugates of 1,2-dihydroxy-1-phenylpropane (31% of the dose) and of 1-hydroxy-1-phenylpropan-2-one (27%) and hippuric acid (20%). The unchanged drug was excreted in relatively small amounts (8%).     

Biodegradation of the herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) by purified pentachlorophenol hydroxylase and whole cells of Flavobacterium sp. strain ATCC 39723 is accompanied by cyanogenesis.

A pentachlorophenol (PCP)-degrading Flavobacterium sp. (strain ATCC 39723) degraded bromoxynil with the production of bromide and cyanide. No aromatic intermediates were detected in the spent culture fluid. The cyanide produced upon bromoxynil metabolism was inhibitory to the Flavobacterium sp. Whole cells degraded PCP more rapidly than they did bromoxynil. Bromoxynil metabolism and PCP metabolism were coinduced, either substrate serving as the inducer. Purified PCP hydroxylase degraded bromoxynil with stoichiometric accumulation of cyanide and without bromide production. A product accumulated which was more hydrophilic than bromoxynil upon high-pressure liquid chromatographic analysis and which, when analyzed by gas chromatography-mass spectrometry, had a mass spectrum consistent with that expected for dibromohydroquinone. PCP hydroxylase consumed NADPH, oxygen, and bromoxynil in a 2:1:1 molar ratio, producing 1 mol of cyanide per mol of bromoxynil degraded. We propose a pathway by which bromoxynil is metabolized by the same enzymes which degrade PCP. The initial step in the pathway is the conversion of bromoxynil to 2,6-dibromohydroquinone by PCP hydroxylase. In addition to its utility for decontaminating PCP-polluted sites, the Flavobacterium sp. may be useful for decontaminating bromoxynil spills. This is the first report of cyanide production accompanying the metabolism of a benzonitrile derivative.     

Biodegradation of the herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) by purified pentachlorophenol hydroxylase and whole cells of Flavobacterium sp. strain ATCC 39723 is accompanied by cyanogenesis.

A pentachlorophenol (PCP)-degrading Flavobacterium sp. (strain ATCC 39723) degraded bromoxynil with the production of bromide and cyanide. No aromatic intermediates were detected in the spent culture fluid. The cyanide produced upon bromoxynil metabolism was inhibitory to the Flavobacterium sp. Whole cells degraded PCP more rapidly than they did bromoxynil. Bromoxynil metabolism and PCP metabolism were coinduced, either substrate serving as the inducer. Purified PCP hydroxylase degraded bromoxynil with stoichiometric accumulation of cyanide and without bromide production. A product accumulated which was more hydrophilic than bromoxynil upon high-pressure liquid chromatographic analysis and which, when analyzed by gas chromatography-mass spectrometry, had a mass spectrum consistent with that expected for dibromohydroquinone. PCP hydroxylase consumed NADPH, oxygen, and bromoxynil in a 2:1:1 molar ratio, producing 1 mol of cyanide per mol of bromoxynil degraded. We propose a pathway by which bromoxynil is metabolized by the same enzymes which degrade PCP. The initial step in the pathway is the conversion of bromoxynil to 2,6-dibromohydroquinone by PCP hydroxylase. In addition to its utility for decontaminating PCP-polluted sites, the Flavobacterium sp. may be useful for decontaminating bromoxynil spills. This is the first report of cyanide production accompanying the metabolism of a benzonitrile derivative.     

The survival of the pentachlorophenol-degrading Rhodococcus chlorophenolicus PCP-1 and Flavobacterium sp. in natural soil

The survival of two different pentachlorophenol (PCP)-degrading bacteria were studied in natural soil. The PCP-degraders Rhodococcus chlorophenolicus and Flavobacterium sp., both able to mineralize PCP into CO₂ and chloride in axenic culture, were tested for the capacity to survive and degrade PCP in natural soil. These bacteria were immobilized on polyurethane (PUR) foam and introduced into natural peaty soil to give about 10⁹ cells g^-1 of soil (dry weight). R. chlorophenolicus induced PCP-degrading activity in soil remained detectable for 200 days whether or not a carbon source was added (distillery waste or wood chips). Electron microscopic investigation performed almost a year after inoculation, revealed the presence of R. chlorophenolicus-like cells in the PUR foam particles. PCP-degrading activity of Flavobacterium sp. declined within 60 days of burial in the soil without enhancing the PCP removal. R. chlorophenolicus degraded PCP in soil at a mean rate of 3.7 mg of PCP day^-1 kg^-1 of soil, which corresponds to ca. 5×10^-3 pg of PCP degraded per inoculated R. chlorophenolicus cell day^-1. The solvent extractable organic chlorine contents of the soil decreased stoichiometrically (>95%) with that of PCP indicating that PCP was essentially mineralized.Abbreviations ATCC American type culture collection - DSM Deutsche Sammlung für Mikroorganismen - DW distillery waste - EM electron microscopy - EOX extractable organic halogen - GC/ECD gas chromatograph/electron capture detector - GC/MS gas chromatograph/mass spectrometer - PCP pentachlorophenol - WC wood chips - d.wt. dry weight - w.wt. wet weight - d.s. dry soil - d.H₂O distilled water - PCA polychlorinated aromatics     

Degradation of pentachlorophenol by polyurethane-immobilized Flavobacterium cells.

Polyurethane-immobilized Flavobacterium cells (ATCC 39723) degraded pentachlorophenol (PCP) at initial concentrations as high as 300 mg liter-1. The reversible binding of PCP to the polyurethane was shown to be important in the protection of the cells from inhibition of PCP degradation. The degradation activity of the bacteria was monitored for 150 days in semicontinuous batch reactors. The degradation rate dropped by about 0.6% per day. PCP was degraded in a continuous-culture bioreactor at a rate of 3.5 to 4 mg g of foam-1 day-1 for 25 days. Electron micrographs of the polyurethane suggested that the cells were entrapped within 50- to 500-microns-diameter pockets in the foam.     

Degradation of pentachlorophenol by polyurethane-immobilized Flavobacterium cells

Polyurethane-immobilized Flavobacterium cells (ATCC 39723) degraded pentachlorophenol (PCP) at initial concentrations as high as 300 mg liter-1. The reversible binding of PCP to the polyurethane was shown to be important in the protection of the cells from inhibition of PCP degradation. The degradation activity of the bacteria was monitored for 150 days in semicontinuous batch reactors. The degradation rate dropped by about 0.6% per day. PCP was degraded in a continuous-culture bioreactor at a rate of 3.5 to 4 mg g of foam-1 day-1 for 25 days. Electron micrographs of the polyurethane suggested that the cells were entrapped within 50- to 500-microns-diameter pockets in the foam.     

Reductive dehalogenation of chlorophenols by Desulfomonile tiedjei DCB-1.

Reductive dehalogenation of chlorophenols has been reported in undefined anaerobic cultures but never before in an anaerobic pure culture. We found that the sulfate-reducing bacterium Desulfomonile tiedjei DCB-1 reductively dehalogenates pentachlorophenol (PCP) and other chlorophenols. The maximum rate of PCP dechlorination observed was 54 mu mol of Cl- h-1 g of protein-1. 3-Chlorobenzoate appeared to serve as a required inducer for PCP dehalogenation; however, neither PCP nor 3-chlorophenol induced dehalogenation. Dehalogenation was catalyzed by living cells, and formate served as a required electron donor. D. tiedjei dehalogenated meta-chlorine substituents of chlorophenols (i.e., PCP was degraded to 2,4,6-trichlorophenol). Generally, more highly chlorinated phenol congeners were more readily dechlorinated, and 3-chlorophenol was not dehalogenated. Growing cultures dehalogenated PCP, but greater than 10 microM PCP (approximately 1.7 mmol g of protein-1) reversibly inhibited growth.     

Reductive dehalogenation of chlorophenols by Desulfomonile tiedjei DCB-1.

Reductive dehalogenation of chlorophenols has been reported in undefined anaerobic cultures but never before in an anaerobic pure culture. We found that the sulfate-reducing bacterium Desulfomonile tiedjei DCB-1 reductively dehalogenates pentachlorophenol (PCP) and other chlorophenols. The maximum rate of PCP dechlorination observed was 54 mu mol of Cl- h-1 g of protein-1. 3-Chlorobenzoate appeared to serve as a required inducer for PCP dehalogenation; however, neither PCP nor 3-chlorophenol induced dehalogenation. Dehalogenation was catalyzed by living cells, and formate served as a required electron donor. D. tiedjei dehalogenated meta-chlorine substituents of chlorophenols (i.e., PCP was degraded to 2,4,6-trichlorophenol). Generally, more highly chlorinated phenol congeners were more readily dechlorinated, and 3-chlorophenol was not dehalogenated. Growing cultures dehalogenated PCP, but greater than 10 microM PCP (approximately 1.7 mmol g of protein-1) reversibly inhibited growth.     

Stability of (1R,2S)-(-)-Ephedrine hydrochloride in Candida albicans-inoculated urine and blood samples

The stability of drugs in biological evidence during collection and storage is of particular concern in forensic investigations. Microbes in the samples and other elements are an essential component of these investigations. In the current study, the HPLC method was used to examine the stability of (1R, 2S)-(-)-Ephedrine hydrochloride in plasma and urine samples inoculated with C. albicans after storage at 37 °C for 48 h and −20 °C for six months. In the stability experiment, MIC_50% of (1R, 2S)-(-)-Ephedrine hydrochloride was applied according to MIC and MFC that were determined in this work. This drug had MIC and MFC of 50 and 100 ppm, respectively. In HPLC analysis, the standard (1R, 2S)-(-)-Ephedrine hydrochloride had a retention time of 1.63 and was used to identify this drug in samples that had or had not been exposed to C. albicans. The findings demonstrated that within 48 h at 37 °C, C. albicans had an impact on the drug concentration (10% and more than 15%, respectively, were lost in plasma and urine samples inoculated with C. albicans). In plasma samples, the drug content remained stable at −20 °C for three months, although it degraded in urine samples after one month. In plasma and urine samples, the concentration reduction had surpassed 70% and 50% by the sixth month, respectively. The results of this investigation show that C. albicans can change the stability of (1R, 2S)-(-)-Ephedrine hydrochloride in plasma and urine samples that contain MIC_50% of Ephedrine hydrochloride.     

Pentachlorophenol degradation by Pseudomonas aeruginosa

Five Pseudomonas species were tested for ability to degrade pentachlorophenol (PCP). Pseudomonas aeruginosa completely degraded PCP up to 800 mg/l in 6 days with glucose as co-substrate. With 1000 mg PCP/l, 53% was degraded. NH₄ ⁺ salts were better at enhancing degradation than organic nitrogen sources and shake-cultures promoted PCP degradation compared with surface cultures. Degradation was maximal at pH 7.6 to 8.0 and at 30 to 37°C. Only PCP induced enzymes that degraded PCP and chloramphenicol inhibited this process. The PCP was degraded to CO₂, with release of Cl^-.The authors are with the Bacteriology Laboratory, Central Leather Research Institute, Madras-600 020, India.     

Characterization of a novel Pseudomonas sp. that mineralizes high concentrations of pentachlorophenol.

A pentachlorophenol (PCP)-mineralizing bacterium was isolated from polluted soil and identified as Pseudomonas sp. strain RA2. In batch cultures, Pseudomonas sp. strain RA2 used PCP as its sole source of carbon and energy and was capable of completely degrading this compound as indicated by radiotracer studies, stoichiometric release of chloride, and biomass formation. Pseudomonas sp. strain RA2 was able to mineralize a higher concentration of PCP (160 mg liter-1) than any previously reported PCP-degrading pseudomonad. At a PCP concentration of 200 mg liter-1, cell growth was completely inhibited and PCP was not degraded, although an active population of Pseudomonas sp. RA2 was still present in these cultures after 2 weeks. The inhibitory effect of PCP was partially attributable to its effect on the growth rate of Pseudomonas sp. strain RA2. The highest specific growth rate (mu = 0.09 h-1) was reached at a PCP concentration of 40 mg liter-1 but decreased at higher or lower PCP concentrations, with the lowest mu (0.05 h-1) occurring at 150 mg liter-1. Despite this reduction in growth rate, total biomass production was proportional to PCP concentration at all PCP concentrations degraded by Pseudomonas sp. RA2. In contrast, final cell density was reduced to below expected values at PCP concentrations greater than 100 mg liter-1. These results indicate that, in addition to its effect as an uncoupler of oxidative phosphorylation, PCP may also inhibit cell division in Pseudomonas sp. strain RA2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a novel Pseudomonas sp. that mineralizes high concentrations of pentachlorophenol.

A pentachlorophenol (PCP)-mineralizing bacterium was isolated from polluted soil and identified as Pseudomonas sp. strain RA2. In batch cultures, Pseudomonas sp. strain RA2 used PCP as its sole source of carbon and energy and was capable of completely degrading this compound as indicated by radiotracer studies, stoichiometric release of chloride, and biomass formation. Pseudomonas sp. strain RA2 was able to mineralize a higher concentration of PCP (160 mg liter-1) than any previously reported PCP-degrading pseudomonad. At a PCP concentration of 200 mg liter-1, cell growth was completely inhibited and PCP was not degraded, although an active population of Pseudomonas sp. RA2 was still present in these cultures after 2 weeks. The inhibitory effect of PCP was partially attributable to its effect on the growth rate of Pseudomonas sp. strain RA2. The highest specific growth rate (mu = 0.09 h-1) was reached at a PCP concentration of 40 mg liter-1 but decreased at higher or lower PCP concentrations, with the lowest mu (0.05 h-1) occurring at 150 mg liter-1. Despite this reduction in growth rate, total biomass production was proportional to PCP concentration at all PCP concentrations degraded by Pseudomonas sp. RA2. In contrast, final cell density was reduced to below expected values at PCP concentrations greater than 100 mg liter-1. These results indicate that, in addition to its effect as an uncoupler of oxidative phosphorylation, PCP may also inhibit cell division in Pseudomonas sp. strain RA2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nicotine metabolism in healthy smokers and patients with cardiovascular diseases

In this study, we measured the excretion rate of nicotine and its two major metabolites, cotinine and trans-3′-hydroxycotinine (THOC), in the urine of 25 healthy smokers and 15 smokers who underwent a coronary artery bypass surgery or coronary angioplasty. After 1 day of smoking cessation, urine samples were collected in the morning, before smoking two cigarettes, and then three times after smoking, approximately 4 h apart. The results show that (i) in healthy smokers, nicotine and its two major metabolites were present at high concentration in the first urine sample after smoking, (ii) in smokers with cardiovascular disease nicotine and cotinine were less excreted whereas THOC was more excreted, mainly in the second urine sample. We conclude that this shift in nicotine metabolism may contribute to smoking-induced cardiovascular disease. (Mol Cell Biochem xxx: 241–244, 2005)     

Effects of Sulfuroxy Anions on Degradation of Pentachlorophenol by a Methanogenic Enrichment Culture

We studied the degradation of pentachlorophenol (PCP) under methanogenic and sulfate-reducing conditions with an anaerobic mixed culture derived from sewage sludge. The consortium degraded PCP via 2,3,4,5-tetrachlorophenol, 3,4,5-trichlorophenol, and 3,5-dichlorophenol and eventually accumulated 3-chlorophenol. Dechlorination of PCP and metabolites was inhibited in the presence of sulfate, thiosulfate, and sulfite. A decrease in the rate of PCP transformation was noted when the endogenous dissolved H₂ was depleted below 0.11 μM in sulfate-reducing cultures. The effect on dechlorination observed with sulfate could be relieved by addition of molybdate, a competitive inhibitor of sulfate reduction. Addition of H₂ reduced the inhibition observed with sulfuroxy anions. The inhibitory effect of sulfuroxy anions may be due to a competition for H₂ between sulfate reduction and dechlorination. When cultured under methanogenic conditions, the consortium degraded several chlorinated and brominated phenols.     

Degradation of pentachlorophenol with the presence of fermentable and non-fermentable co-substrates in a microbial fuel cell

Pentachlorophenol (PCP) was more rapidly degraded in acetate and glucose-fed microbial fuel cells (MFCs) than in open circuit controls, with removal rates of 0.12 ± 0.01 mg/Lh (14.8 ± 1.0 mg/g-VSS-h) in acetate-fed, and 0.08 ± 0.01 mg/L h (6.9 ± 0.8 mg/g-VSS-h) in glucose-fed MFCs, at an initial PCP concentration of 15 mg/L. A PCP of 15 mg/L had no effect on power generation from acetate but power production was decreased with glucose. Coulombic balances indicate the predominant product was electricity (16.1 ± 0.3%) in PCP-acetate MFCs, and lactate (19.8 ± 3.3%) in PCP-glucose MFCs. Current generation accelerated the removal of PCP and co-substrates, as well as the degradation products in both PCP-acetate and PCP-glucose reactors. While 2,3,4,5-tetrachlorophenol was present in both reactors, tetrachlorohydroquinone was only found in PCP-acetate MFCs. These results demonstrate PCP degradation and power production were affected by current generation and the type of electron donor provided.     

Self-titration by cigarette smokers

An 11-week crossover study was carried out in which 12 subjects smoked high-nicotine (1·84 mg standard yield) and low-nicotine (0·6 mg) cigarettes after an initial period of smoking their usual brands with a medium-nicotine yield (mean 1·4 mg). Plasma and urine nicotine concentrations, carboxyhaemoglobin (COHb) concentration, puffing behaviour, 24-hour cigarette consumption, and butt nicotine content were measured. The changes in plasma nicotine and blood COHb concentrations showed that the smokers compensated for about two-thirds of the difference in standard yields when switched to either high- or low-nicotine cigarettes. Thus, compared with the medium-nicotine brand, the intake of nicotine and carbon monoxide was only about 10% higher when subjects smoked the high-nicotine cigarettes, which had a standard yield 30-40% higher than the medium brands; and only about 15% lower when they smoked the low-nicotine cigarettes, which had a standard yield about 50% lower than the medium brands. But nicotine content and urine nicotine concentrations followed a similar pattern. Changes in puffing behaviour and in 24-hour cigarette consumption were only slight.The results show clear evidence of both upward and downward self-titration of nicotine and carbon monoxide (and tar) intakes when smokers change to cigarettes with standard yields that differ over the range studied.