Degradation of 5-chloro-2-hydroxynicotinic acid by Mycobacterium sp. BA

Bacteria with the ability to use 5-chloro-2-hydroxynicotinic acid as sole source of carbon and energy have been isolated from enrichment cultures with 2-hydroxynicotinic acid and 5-chloro-2-hydroxynicotinic acid. According to their morphological and physiological properties, these bacteria have been classified as Mycobacterium sp. The first metabolite in the degradation pathway is 5-chloro-2,6-dihydroxynicotinic acid. 2-Hydroxynicotinic acid had an inductive effect on the degrading enzymes. Chloride was released from 5-chloro-2-hydroxynicotinic acid, chloromaleic acid and chlorofumaric acid. A degradation pathway is proposed.     

Biodegradation of 5-chloro-2-picolinic acid by novel identified co-metabolizing degrader <Emphasis Type="Italic">Achromobacter</Emphasis> sp. f1

Several bacteria have been isolated to degrade 4-chloronitrobenzene. Degradation of 4-chloronitrobenzene by Cupriavidus sp. D4 produces 5-chloro-2-picolinic acid as a dead-end by-product, a potential pollutant. To date, no bacterium that degrades 5-chloro-2-picolinic acid has been reported. Strain f1, isolated from a soil polluted by 4-chloronitrobenzene, was able to co-metabolize 5-chloro-2-picolinic acid in the presence of ethanol or other appropriate carbon sources. The strain was identified as Achromobacter sp. based on its physiological, biochemical characteristics, and 16S rRNA gene sequence analysis. The organism completely degraded 50, 100 and 200 mg L^?1 of 5-chloro-2-picolinic acid within 48, 60, and 72 h, respectively. During the degradation of 5-chloro-2-picolinic acid, Cl^? was released. The initial metabolic product of 5-chloro-2-picolinic acid was identified as 6-hydroxy-5-chloro-2-picolinic acid by LC–MS and NMR. Using a mixed culture of Achromobacter sp. f1 and Cupriavidus sp. D4 for degradation of 4-chloronitrobenzen, 5-chloro-2-picolinic acid did not accumulate. Results infer that Achromobacter sp. f1 can be used for complete biodegradation of 4-chloronitrobenzene in remedial applications.     

Phytotoxic Metabolites of Phyllosticta sp.

The additional new metabolites, named phyllostine (II) and 3-chloro-2,5-dihydroxybenzyl alcohol (III) from the cultural filtrates of Phyllosticta sp. have been isolated. The chemical structure of II have been established by spectral studies and chemical conversion from the known I, and the chlorine-containing metabolite (III) by spectral and synthetic studies. The metabolite (II) exhibits the similar phytotoxic effects as I, but the metabolite III does less phytotoxicity than I and II on the leaf test.     

Metabolism of 4-chloro-2-methylphenoxyacetate by a soil pseudomonad. Preliminary evidence for the metabolic pathway

1. A pseudomonad capable of utilizing the herbicide 4-chloro-2-methylphenoxyacetate as a sole carbon source was isolated from soil and cultured in liquid medium. 2. Analysis of induction patterns of 4-chloro-2-methylphenoxyacetate-grown cells suggests that 5-chloro-o-cresol and 5-chloro-3-methylcatechol are early intermediates in the oxidation of 4-chloro-2-methylphenoxyacetate. Cells were not adapted to oxidize 4-chloro-6-hydroxy-2-methylphenoxyacetate. 3. In culture, 4-chloro-2-methylphenoxyacetate rapidly disappeared and the chlorine in the molecule was quantitatively released as Cl(-) ion. 4. A lactone (gamma-carboxymethylene-alpha-methyl-Delta(alphabeta)-butenolide) was isolated from cultures and established as an intermediate. 5. The following metabolic pathway is suggested: 4-chloro-2-methylphenoxyacetate --> 5-chloro-o-cresol --> 5-chloro-3-methylcatechol --> cis-cis-gamma-chloro-alpha-methylmuconate --> gamma-carboxymethylene-alpha-methyl-Delta(alphabeta)-butenolide --> gamma-hydroxy-alpha-methylmuconate. 6. The tentative identification of 5-chloro-o-cresol, a gamma-chloro-alpha-methylmuconate and gamma-hydroxy-alpha-methylmuconate in culture extracts supports this scheme. However, the catechol was never observed to accumulate in cultures. 7. The detection of 4-chloro-6-hydroxy-2-methylphenoxyacetate, 2-methyl-phenoxyacetate, a dehalogenated cresol and oxalate in culture extracts is discussed in relation to the proposed metabolic pathway.     

Studies on plant growth-regulating substances. XL. 2-Chloro-3-phenylpropionitriles

The complete series of chloro-, dichloro-, methyl-, dimethyl- and chloro-methyl-ring substituted 2-chloro-3-phenylpropionitriles and several related compounds have been prepared. Their auxin activities have been assessed in the wheat coleoptile, pea segment and pea curvature tests, and the results, which are discussed in relation to molecular structure, support the suggestion by other workers that the observed activity is due to rapid uptake of the nitriles by the plant tissues, followed by metabolism to the corresponding phenylacetic acid. The high growth promoting activity of 2-chloro-3-(3-chloro-2-methyl-phenyl)propionitrile (orthonil) is confirmed but 2-chloro-3-(2,3-dichloro-phenyl)propionitrile and 2-chloro-3-(2,3-dichlorophenyl)butyronitrile are shown to be the most active members of the series.     

The degradation of cholic acid by Pseudomonas sp. N.C.I.B. 10590 under anaerobic conditions.

The bacterial degradation of cholic acid under anaerobic conditions by Pseudomonas sp. N.C.I.B. 10590 was studied. The major unsaturated neutral compound was identified as 12 beta-hydroxyandrosta-4,6-diene-3,17-dione, and the major unsaturated acidic metabolite was identified as 12 alpha-hydroxy-3-oxochola-4,6-dien-24-oic acid. Eight minor unsaturated metabolites were isolated and evidence is given for the following structures: 12 alpha-hydroxyandrosta-4,6-diene-3,17-dione, 12 beta,17 beta-dihydroxyandrosta-4,6-dien-3-one, 12 beta-hydroxyandrosta-1,4,6-triene-3,17-dione, 12 beta,17 beta-dihydroxyandrosta-1,4,6-trien-3-one, 12 beta-hydroxyandrosta-1,4,6-triene-3,17-dione, 12 beta,17 beta-dihydroxyandrosta-1,4,6-trien-3-one, 12 alpha-hydroxyandrosta-1,4-diene-3,17-dione, 3-hydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione, 3,12-dioxochola-4,6-dien-24-oic acid and 12 alpha-hydroxy-3-oxopregna-4,6-diene-20-carboxylic acid. In addition, a major saturated neutral compound was isolated and identified as 3 beta,12 beta-dihydroxy-5 beta-androstan-17-one, and the only saturated acidic metabolite was 7 alpha,12 alpha-dihydroxy-3-oxo-5 beta-cholan-24-oic acid. Nine minor saturated neutral compounds were also isolated, and evidence is presented for the following structures: 12 beta-hydroxy-5 beta-androstane-3,17-dione, 12 alpha-hydroxy-5 beta-androstane-3,17-dione, 3 beta,12 alpha-dihydroxy-5 beta-androstan-17-one, 3 alpha,12 beta-androstan-17-one, 3 alpha,12 alpha-dihydroxy-5 beta-androstan-17-one, 5 beta-androstane-3 beta,12 beta,17 beta-triol, 5 beta-androstane-3 beta,12 alpha,17 beta-triol, 5 beta-androstane-3 alpha,12 beta,17 beta-triol and 5 beta-androstane-3 alpha,12 alpha,17 beta-triol. The induction of 7 alpha-dehydroxylase and 12 alpha-dehydroxylase enzymes is discussed, together with the significance of dehydrogenation and ring fission under anaerobic conditions.     

Synthesis of chloromethyl ketone derivatives of fatty acids. Their use as specific inhibitors of acetoacetyl-coenzyme A thiolase, cholesterol biosynthesis and fatty acid synthesis.

A general route for the synthesis of chloromethyl ketone derivatives of fatty acids is described. 5-Chloro-4-oxopentanoic acid, 7-chloro-6-oxoheptanoic acid, 9-chloro-8-oxononanoic acid and 11-chloro-10-oxoundecanoic acid were synthesized by this method and tested as covalent inhibitors of pig heart acetoacetyl-CoA thiolase. The K1 decreased by approx. 20-fold for each pair of methylenes added to the chain length, showing that the initial stage in inhibitor binding occurs at a non-polar region of the protein. This region is probably located at the enzyme active site, since inhibition was prevented by acetoacetyl-CoA or acetyl-CoA but not by CoA. The site of modification by chloromethyl ketone derivatives of fatty acids is restricted to a thiol group, since inactivation of the enzyme was prevented by reversible thiomethylation of the active-site thiol. In contrast, an amino-directed reagent, citraconic anhydride, still inactivated the enzyme, even when the active-site thiol was protected. Evidence that the enzyme thiol was particularly reactive came from studies on the pH-dependence of the alkylation reaction and thiol-competition experiments. Inhibition of the enzyme proceeded suprisingly well at acidic pH values and a 10(5) molar excess of external thiol over active-site thiol was required to prevent inhibition by 0.3 mM-9-chloro-8-oxononanoic acid. In addition to inhibiting isolated acetoacetyl-CoA thiolase, in hepatocytes the chloromethyl ketone derivatives of fatty acids also inhibited chloresterol synthesis, which uses this enzyme as an early step in the biosynthetic pathway. In isolated cells, the chloromethyl ketone derivatives of fatty acids were considerably less specific in their inhibitory action compared with 3-acetylenic derivatives of fatty acids, which act as suicide inhibitors of acetoacetyl-CoA thiolase. However, 9-chloro-8-oxononanoic acid was also an effective inhibitor of both hepatic cholesterol and fatty acid synthesis in mice in vivo, whereas the acetylenic fatty acid derivative, dec-3-ynoic acid, was completely ineffective. The effective inhibitory dose of 9-chloro-8-oxononanoic acid (2.5-5 mg/kg) was substantially lower than the estimated LD50 for the inhibitor (100 mg/kg).     

Metabolism of dibenzothiophene by a Beijerinckia species.

Beijerinckia B8/36 when grown with succinate in the presence of dibenzothiophene, accumulated (+)-cis-1,2-dihydroxy-1,2-dihydrodibenzothiophene and dibenzothiophene-5-oxide in the culture medium. Each metabolite was isolated in crystalline form and characterized by a variety of chemical techniques, cis-Naphthalene dihydrodiol dehydrogenase, isolated from Pseudomonas putida, oxidized (+)-cis-1,2-dihydroxy-1,2-dihydrodibenzothiophene to a compound that was tentatively identified as 1,2-dihydroxydibenzothiophene. The same product was formed when crude cell extracts of the parent strain of Beijerinckia oxidized (+)-cis-1,2-dihydroxy-1,2-dihydrodibenzothiophene under anaerobic conditions. Further metabolism of 1,2-dihydroxydibenzothiophene by heat-treated cell extracts led to the formation of 4[2-(3-hydroxy)-thionaphthenyl]-2-oxo-3-butenoic acid. The latter compound was metabolized by crude cell extracts to 3-hydroxy-2-formylthionaphthene. Further degradation of this metabolite was not observed.     

R-stereoselective amidase from Rhodococcus erythropolis No. 7 acting on 4-chloro-3-hydroxybutyramide

Ethyl (S)-4-chloro-3-hydroxybutyrate is an intermediate for the synthesis of Atorvastatin, a chiral drug used for hypercholesterolemia. A Rhodococcus erythropolis strain (No. 7) able to convert 4-chloro-3-hydroxybutyronitrile into 4-chloro-3-hydroxybutyric acid has recently been isolated from soil. This activity has been regarded as having been caused by the successive actions of the nitrile hydratase and amidase. In this instance, the corresponding amidase gene was cloned from the R. erythropolis strain and expressed in Escherichia coli cells. A soluble active form of amidase enzyme was obtained at 18 degrees . The Ni column-purified recombinant amidase was found to have a specific activity of 3.89 U/mg toward the substrate isobutyramide. The amidase was found to exhibit a higher degree of activity when used with midchain substrates than with short-chain ones. Put differently, amongst the various amides tested, isobutyramide and butyramide were found to be hydrolyzed the most rapidly. In addition to amidase activity, the enzyme was found to exhibit acyltransferase activity when hydroxyl amine was present. This dual activity has also been observed in other enzymes belonging to the same amidase group (E.C. 3.5.1.4). Moreover, the purified enzyme was proven to be able to enantioselectively hydrolyze 4-chloro-3-hydroxybutyramide into the corresponding acid. The e.e. value was measured to be 52% when the conversion yield was 57%. Although this e.e. value is low for direct commercial use, molecular evolution could eventually result in this amidase being used as a biocatalyst for the production of ethyl (S)-4-chloro-3-hydroxybutyrate.     

Isolation and characterization of a 3-chlorobenzoate degrading pseudomonad

A pseudomonad has been isolated from sewage, which can utilize 3-chlorobenzoic acid as a sole carbon source. In cells grown on benzoate the enzymes of the -ketoadipic acid pathway are present. Considerable enzymic activities for chlorinated substrates were found in benzoate grown cells only for the oxygenation of 3-chlorobenzoate and the dehydrogenation of 3- and 5-chloro-3,5-cyclohexadiene-1,2-diol-1-carboxylic acid. 3-Chlorobenzoate grown cells show additional high activities for the turnover of 3- and 4-chlorocatechols and chloromuconic acids.Abbreviations Used DHB (-)-3,5-cyclohexadiene-1,2-diol-1-carboxylic acid (derived from the trivial name, dihydrodihydroxybenzoate) - 3- and 5-Cl-DHB correspondingly 3- and 5-chloro-3,5-cyclohexadiene-1,2-diol-1-carboxylic acid     

Microbiological degradation of bile acids, further degradation of a cholic acid metabolite containing the hexahydroindane nucleus by Corynebacterium equi.

1. The further degradation of a cholic acid (I) metabolite, (4R)-4-[4alpha-(2-carboxyethyl)-3aalpha-hexahydro-7abeta-methyl-5-oxoindan-1beta-yl]valeric acid (IIa), by Corynebacterium equi was investigated. This organism effected ring-opening and gave (4R)-4-[2alpha-(2-carboxyethyl)-3beta-(3-carboxypropionyl)-2beta-methylcyclopent-1beta-yl]valeric acid (VI). The new metabolite was isolated as its trimethyl ester and identified by partical synthesis. It was not utilized by C. equi. 2. (4R)-4[4alpha-(2-Carboxyethyl)-3aalpha-decahydro-8abeta-methyl5-oxa-6-oxoazulen-1beta-yl]valeric acid (IVa), which is a hypothetical initial oxidation product in the above degradation, was not converted by C. equi into the expected metabolite (VI), but into 3 - [2beta - [(2S) - tetrahydro - 5 - oxofur - 2 - yl] - 1beta - methyl - 5 - oxocyclopent - 1alpha - yl]-propionic acid (VIII), the structure of which was established by partial synthesis. 3. Both the possible precursors of the metabolite (VI), an isomer of the epsilon-lactone (IVa), the gamma-lactone (XIa), and the open form of these lactones, the hydroxytricarboxylic acid (V), were also not utilized by C. equi. 4. Under some incubation conditions, C. equi also converted compound (IIa) and 3-(3aalpha-hexahydro-7abeta-methyl-1,5-dioxoindan-4alpha-yl)propionic acid (IIb) into 5-methyl-4-oxo-octane-1,8-dioic acid (III), (4R)-4-(2,3,4,6,6abeta,7,8,9,9aalpha,9bbeta-decahydro-6abeta-methyl-3-oxo-1H-cyclopenta[f]quinolin-7beta-yl)valeric acid (VII) and probably a monohydroxy derivative of compound (IIa) and compound (III), respectively. 5. The possibility that an initial step in the degradation of compound (IIa) by C. equi is oxygenation of the Baeyer-Villiger type, yielding compound (IVa), is discussed. Metabolic pathways of compound (IIa) to compounds (III), (VI), (VII) and (VIII) are also considered.     

Enzymatic and chemical preparation of 5-amino-4-chloro-2-(2,3-dihydroxyphen-1-yl)-3(2H)-pyridazinone

A hypothetical intermediate of the microbial degradation of pyrazon, 5-amino-4-chloro-2(2,3-dihydroxyphen-1-yl)-3(2H)-pyridazinone, was prepared by enzymatic and chemical treatment of 5-amino-4-chloro-2(2,3-dihydroxy-cyclohexa-4,6-dien-1-yl)-pyridazinone. The properties of the metabolite are described.     

Selective modification of rabbit muscle pyruvate kinase by 5-chloro-4-oxopentanoic acid.

Rabbit muscle pyruvate kinase was irreverisbly inactivated by 5-chloro-4-oxopentanoic acid with a pKa of 9.2. The inhibition was time-dependent and was related to the 5-chloro-4-oxopentanoic acid concentration. Analysis of the kinetics of inhibition showed that the binding of the inhibitor showed positive co-operativity (n = 1.5 +/- 0.2). Inhibition of pyruvate kinase by 5-chloro-4-oxopentanoic acid was prevented by ligands which bind to the active site. Their effectiveness was placed in the order Mg2+ greater than phosphoenolpyruvate greater than ATP greater than ADP greater than pyruvate. Inhibitor-modified pyruvate kinase was unable to catalyse the detritiation of [3-(3)H]pyruvate in the ATP-promoted reaction, but it did retain 5-10% of the activity with either phosphate or arsenate as promoters. 5-Chlor-4-oxo-[3,5-(3)H]pentanoic acid was covalently bound to pyruvate kinase and demonstrated a stoicheiometry of 1 mol of inhibitor bound per mol of pyruvate kinase protomer. The incorporation of the inhibitor and the loss of enzyme was proportional. These results are discussed in terms of 5-chloro-4-oxopentanoic acid alkylating a functional group in the phosphoryl overlap region of the active site, and a model is presented in which this compound alkylates an active-site thiol in a reaction that is controlled by a more basic group at the active site.     

Isolation and Characterization of a Novel Atrazine Metabolite Produced by the Fungus Pleurotus pulmonarius, 2-Chloro-4-Ethylamino-6-(1-Hydroxyisopropyl)Amino-1,3,5-Triazine

The white rot fungus Pleurotus pulmonarius exhibited metabolism of atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine) in liquid culture, producing the dealkylated metabolites desethylatrazine, desisopropylatrazine, and desethyl-desisopropylatrazine. A fourth, unknown metabolite was also produced. It was isolated and was identified as 2-chloro-4-ethylamino-6-(1-hydroxyisopropyl)amino-1,3,5-triazine by gas chromatography-mass spectrometry, Fourier transformed infrared spectroscopy, and ¹H nuclear magnetic resonance analysis. The structure of this metabolite was confirmed by chemical synthesis of the compound and comparison with the fungally produced metabolite.     

Effects of hydroxylated polychlorinated biphenyls on mouse liver mitochondrial oxidative phosphorylation.

The effects of three tetrachlorobiphenylols [2',3',4',5'-tetrachloro-2-biphenylol (1); 2',3',4',5'-tetrachloro-4- biphenylol (2); and 2',3',4',5'-tetrachloro-3-biphenylol (3)]; three monochlorobiphenylols [5-chloro-2-biphenylol (5), 3-chloro-2-biphenylol (6); and 2-chloro-4-biphenylol (7)] and a tetrachlorobiphenyldiol [3,3',5,5'-tetrachloro-4,4'-biphenyldiol (4) on respiration, adenosine triphosphatase (ATPase) activity, and swelling in isolated mouse liver mitochondria have been investigated. Tetrachlorobiphenylols (1-3) and the tetrachlorobiphenyldiol (4) inhibited state-3 respiration in a concentration-dependent manner with succinate as substrate (flavin adenine dinucleotide [FAD]-linked) and the tetrachlorobiphenyldiol (4) caused a more pronounced inhibitory effect on state-3 respiration than the other congeners. The monochlorobiphenylols 5-7 were less active as inhibitors of state-3 mitochondrial respiration and significant effects were observed only at higher concentration (greater than or equal to 0.4 microM). However, in the presence of the nicotinamide adenine dinucleotide (NAD)-linked substrates (glutamate plus malate), hydroxylated PCBs (1-7) significantly inhibited mitochondrial state-3 respiration in a concentration-dependent manner. Compounds 5, 6, and 7 uncoupled mitochondrial oxidative phosphorylation only in the presence of FAD-linked substrate as evidenced by increased oxygen consumption during state-4 respiratory transition, stimulating ATPase activity, releasing oligomycin-inhibited respiration, and inducing mitochondrial swelling (5, 6, and 7). Tetrachlorobiphenylols 1, 2, and 3 had no effect on mitochondrial ATPase activity while the tetrachlorobiphenyldiol, 4, decreased the enzyme activity. The possible inhibitory site of electron transport by these compounds and their toxicologic significance is discussed.     

The metabolism of the isomeric tert.-butylcyclohexanones

1. (+/-)-2-, (+/-)-3- and 4-tert.-Butylcyclohexanone are reduced in the rabbit to secondary alcohols, which are excreted extensively conjugated with glucuronic acid. 2. The major metabolite of (+/-)-2-tert.-butylcyclohexanone is (+)-cis-2-tert.-butylcyclohexanol, which has been isolated from the urine as [(+)-cis-2-tert.-butylcyclohexyl beta-d-glucosid]uronic acid. The minor metabolite is (+)-trans-2-tert.-butylcyclohexanol. 3. (+/-)-3-tert.-Butylcyclohexanone is reduced mainly to (+/-)-cis-3-tert.-butylcyclohexanol, and to a smaller extent to (+/-)-trans-3-tert.-butylcyclohexanol. 4. 4-tert.-Butylcyclohexanone yields mainly the trans-alcohol, which is excreted in conjugated form and has been recovered from the urine as (trans-4-tert.-butylcyclohexyl beta-d-glucosid)uronic acid. The cis-alcohol is formed to a minor extent and excreted in conjugated form. 5. The ratios of the amounts of cis- to trans-alcohols produced by the three ketones differed from the relative amounts of cis- and trans-alcohols produced by the corresponding methylcyclohexanones. 6. From these findings the suggestion is made that two orientations of ketone relative to coenzyme occur: alcohols with an equatorially orientated hydroxyl group are thought to be produced as a result of a ;face-to-face' interaction with NADH, and alcohols with axially orientated hydroxyl groups as a result of a ;perpendicular' interaction. Which will predominate is thought to depend on steric factors, particularly the size and position of alkyl substituents in the substrate.     

Formation of Thiazolidine-4-Carboxylic Acid Represents a Main Metabolic Pathway of 5-Hydroxytryptamine in Rat Brain

Incubation of 5-hydroxytryptamine (5-HT) with rat brain homogenate resulted in the formation of (4R)-2-[3'-(5'-hydroxyindolyl)-methyl]-1,3-thiazolidine-4-carboxyl ic acid (5'-HITCA) as the major metabolite. The substance represents the condensation product of 5-hydroxyindole-3-acetaldehyde with L-cysteine. The chemical structure was confirmed by chromatographic and chemical methods as well as by fast atom bombardment mass spectrometry. Incubation of 5-HT in the presence of L-cysteine yielded the thiazolidine as the main metabolite up to 4 h. Under these conditions, the concentration of 5-hydroxyindole-3-acetic acid (5-HIAA) amounted to about 20% and 57% of 5'-HITCA (0.5 h and 4 h, respectively). In contrast to these findings, indole-3-acetic acid (IAA) was identified as the major metabolite when tryptamine was incubated under similar conditions. (4R)-2-(3'-Indolylmethyl)-1,3-thiazolidine-4-carboxylic acid (ITCA) was found to be the main conversion product of tryptamine only during the first 30 min. To investigate the fate of the thiazolidines, radiolabelled and unlabelled ITCA was incubated with rat brain homogenate. The compound was degraded enzymatically and rapidly. Subcellular fractionation revealed that the enzyme activity was present mainly in the cytosolic fraction whereas the preparation of mitochondria showed less activity. The responsible enzyme is presumably a carbon-sulfur lyase (EC 4.4.1.-). The major metabolite was isolated by HPLC and identified by mass spectrometry as well as by comparison with reference compounds to be IAA.(ABSTRACT TRUNCATED AT 250 WORDS)     

The metabolism and molecular toxicology of chloroprene

Chloroprene (2-chloro-1,3-butadiene, 1) is oxidised by cytochrome P450 enzymes in mammalian liver microsomes to several metabolites, some of which are reactive towards DNA and are mutagenic. Much less of the metabolite (1-chloroethenyl)oxirane (2a/2b) was formed by human liver microsomes compared with microsomes from Sprague-Dawley rats and B6C3F1 mice. Epoxide (2a/2b) was a substrate for mammalian microsomal epoxide hydrolases, which showed preferential hydrolysis of the (S)-enantiomer (2b). The metabolite 2-chloro-2-ethenyloxirane (3a/3b) was rapidly hydrolysed to 1-hydroxybut-3-en-2-one (4) and in competing processes rearranged to 1-chlorobut-3-en-2-one (5) and 2-chlorobut-3-en-1-al (6). The latter compound isomerised to (Z)-2-chlorobut-2-en-1-al (7). In microsomal preparations from human, rat and mouse liver, compounds 4, 5 and 7 were conjugated by glutathione both in the absence and presence of glutathione transferases. There was no evidence for the formation of a chloroprene diepoxide metabolite in any of the microsomal systems. The major adducts from the reaction of (1-chloroethenyl)oxirane (2a/2b) with calf thymus DNA were identified as N7-(3-chloro-2-hydroxy-3-buten-1-yl)-guanine (20) and N3-(3-chloro-2-hydroxy-3-buten-1-yl)-2'-deoxyuridine (23), with the latter being derived by alkylation at N-3 of 2'-deoxycytidine, followed by deamination. Adducts in DNA were identified by comparison with those derived from individual deoxyribonucleosides. The metabolite (Z)-2-chlorobut-2-en-1-al (7) formed principally two adducts with 2'-deoxyadenosine which were identified as a pair of diastereoisomers of 3-(2'-deoxy-beta-d-ribofuranosyl)-7-(1-hydroxyethyl)-3H-imidazo[2,1-i]purine (25). The chlorine atom of chloroprene thus leads to different intoxication and detoxication profiles compared with those for butadiene and isoprene. The results infer that in vivo oxidations of chloroprene catalysed by cytochrome P450 are more important in rodents, whereas hydrolytic processes catalysed by epoxide hydrolases are more pronounced in humans. The reactivity of chloroprene metabolites towards DNA is important for the toxicology of chloroprene, especially when detoxication is incomplete.     

Characterization of Fluoroglycofen Ethyl Degradation by Strain <Emphasis Type="Italic">Mycobacterium phocaicum</Emphasis> MBWY-1

A fluoroglycofen ethyl-degrading bacterium, MBWY-1, was isolated from the soil of an herbicide factory. This isolated strain was identified as Mycobacterium phocaicum based on analysis of its 16S rRNA gene sequence and its morphological, physiological, and biochemical properties. The strain was able to utilize fluoroglycofen ethyl as its sole source of carbon for growth and could degrade 100 mg l⁻¹ of fluoroglycofen ethyl to a non-detectable level within 72 h. The optimum temperature and pH for fluoroglycofen ethyl degradation by strain MBWY-1 were 30°C and 7.0, respectively. Five metabolites produced during the degradation of fluoroglycofen ethyl and were identified by mass spectrometry as {5-[2-chloro-4-(trifluoromethyl) phenoxy]-2-nitrophenylacyl} hydroxyacetic acid, acifluorfen, 5-[2-chloro-4-(trifluoromethyl) phenoxy]-2-nitrobenzoate, 5-[2-chloro-4-(trifluoromethyl) phenoxy]-2-hydroxyl, and 3-chloro-4-hydroxyl benzotrifluoride. Identification of the metabolites allowed to propose the degradation pathway of fluoroglycofen ethyl by strain MBWY-1. The inoculation of strain MBWY-1 into soil treated with fluoroglycofen ethyl resulted in a higher fluoroglycofen ethyl degradation rate than in uninoculated soil regardless of whether the soil was sterilized or nonsterilized.