Assessment of the in vivo genotoxicity of isomers of dinitrotoluene using the alkaline Comet and peripheral blood micronucleus assays

Dinitrotoluene (DNT) is a nitroaromatic explosive that exists as six isomers; two major isomers (2,4- and 2,6-DNT) and four minor isomers (2,3-, 2,5-, 3,4-, and 3,5-DNT). DNT has been found in soil, surface water, and groundwater near ammunition production plants. The major isomers of DNT are classified as "likely to cause cancer in humans."In vitro studies have provided conflicting data regarding the genotoxicity of the minor isomers. Studies indicate that metabolism in the gut and liver are necessary to convert DNT to genotoxic compounds. As such, in the present study the genotoxicity of isomers of DNT was assessed using two in vivo genotoxicity assays. The Comet assay was used to detect DNA damage in liver cells from male Sprague-Dawley rats following oral exposure (14-day) to individual isomers of DNT. The micronucleus assay was conducted using flow cytometric analysis to detect chromosomal damage in peripheral blood. Treatment with 2,3-, 3,4-, 2,4-, 2,5- and 3,5-DNT did not induce DNA damage in liver cells or increase the frequency of micronucleated reticulocytes (MN-RET) in peripheral blood at the doses tested. Treatment with 2,6-DNT induced DNA damage in liver tissue at all doses tested, but did not increase the frequency of micronucleated reticulocytes (MN-RET) in peripheral blood. Thus, 2,4-DNT and the minor isomers were not genotoxic under these test conditions, while 2,6-DNT was genotoxic in the target tissue, the liver. These results support previous research which indicated that the hepatocarcinogenicity of technical grade DNT (TG-DNT) could be attributed to the 2,6-DNT isomer.     

Formation of Dimethylmuconolactones from Dimethylphenols by Alcaligenes eutrophus JMP 134

2,3-, 2,4-, 2,5-, 3,4-, and 3,5-dimethylphenols were cometabolized by 2,4-dichlorophenoxyacetate-grown Alcaligenes eutrophus JMP 134 or the constitutive derivative JMP 134-1 via the ortho pathway into dimethylmuconolactones as dead-end products. Formation of two distinct lactones from 3,4-dimethylphenol is indicative of 2- as well as 6-hydroxylation. Induction of the meta-cleavage pathway by 2,3- and 3,4-dimethylphenols resulted in growth and no accumulation of products. In contrast, 3,5-dimethylphenol is not metabolized by the meta-cleavage pathway.     

Alternative pathways foro-xylene orm-xylene andp-xylene degradation in aPseudomonas stutzeri strain

Pseudomonas stutzeri OX1 is able to grow ono-xylene but is unable to grow onm-xylene andp-xylene, which are partially metabolized through theo-xylene degradative pathway leading to the formation of dimethylphenols toxic to OX1.P. stutzeri spontaneous mutants able to grow onm-xylene andp-xylene have been isolated. These mutants soon lose the ability to grow ono-xylene. Data from HPLC analyses and from induction studies suggest that in these mutantsm-xylene andp-xylene could be metabolized through the oxidation of a methyl substituent.P. stutzeri chromosomal DNA is shown to share homology with pWW0 catabolic genes. In the mutant strains the region homologous to pWW0 upper pathway genes has undergone a genomic rearrangement.Abbreviations BADH benzylalcohol dehydrogenase - cat catechol - C23O catechol 2,3-dioxygenase - 2,3-,3,4-,2,4-,2,6-,3,5-2,5-DMP 2,3-,3,4-,2,4-,2,6-,3,5-,2,5-dimethylphenol - 2-MBOH 2-methylbenzyl alcohol - 3-MBOH 3-methylbenzyl alcohol - 4-MBOH 4-methylbenzyl alcohol - m-,p-tol m-,p-toluate - o-,m-,p-xyl o-,m-,p-xylene     

The suitability of different DHB isomers as matrices for the MALDI-TOF MS analysis of phospholipids: which isomer for what purpose?

Although the analysis of large biomolecules is the prime application of matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS), there is also increasing interest in lipid analysis. Since lipids possess relatively small molecular weights, matrix signals should be as small as possible to avoid overlap with lipid peaks. Although 2,5-dihydroxybenzoic acid (DHB) is an established MALDI matrix, the question whether just this isomer is ideal for lipid analysis was not yet addressed. UV absorptions of all six DHB isomers were determined and their laser desorption spectra recorded. In addition, all isomers were used as matrices to record positive and negative ion mass spectra of selected phospholipids (phosphatidylcholine and -serine): In the order 2,5-, 2,6-, 2,3- and 2,4-DHB, the quality of the positive ion lipid spectra decreases. This correlates well with the decreasing acidity of the applied DHB isomers. The 3,4- and 3,5- isomers give only very weak positive ion signals especially of acidic lipids. In contrast, the most suitable matrices in the negative ion mode are 2,5-, 2,4- and 3,5-DHB. 2,6-DHB does not provide any signal in the negative ion mode due to its marked acidity. Finally, differences in the crystallization behavior of the pure matrix and the matrix/lipid co-crystals were also monitored by atomic force microscopy (AFM): 2,5-DHB gave the smallest crystals and the skinniest layer. It is concluded that basically all DHB isomers can be used as MALDI matrices but the 2,5-isomer represents the most versatile compound. Dedicated to Prof. Dr. Klaus Arnold on the occasion of his 65th birthday.     

Effect of different isomers of dihydroxybenzoic acids (DBA) on the rate of DL-dopa oxidation by mushroom tyrosinase.

Dihydroxybenzoic acids (DBA), such as 3,4-DBA, 3,5-DBA, and 2,4-DBA--at all concentrations tested--inhibited the rate of DL-DOPA oxidation to dopachrome (lambda max = 475 nm) by mushroom tyro0sinase. 2,3-DBA and 2,5-DBA at relatively low concentration had a synergistic effect on the reaction, whereas at relatively high concentrations they inhibited the rate of DL-DOPA oxidation. The synergistic effect of 0.6-13.3 mM 2,3-DBA on the rate of DL-DOPA oxidation to dopachrome (lambda max = 475 nm) was found to be due to the ability of 2,3-DBA-o-quinone (formed by the oxidation of 2,3-DBA by mushroom tyrosinase or by sodium periodate) to oxidize DL-DOPA to dopachrome (via dopaquinone) non-enzymatically. A similar explanation is likely to be valid for the synergism exerted by 2,5-DBA on the rate of DL-DOPA oxidation by mushroom tyrosinase.     

Effect of Different Isomers of Dihydroxybenzoic Acids (DBA) on the Rate of DL-DOPA Oxidation by Mushroom Tyrosinase

Dihydroxybenzoic acids (DBA), such as 3,4-DRA, 3,5-DBA, and 2,4-DBA—at all concentrations tested—inhibited the rate of DL-DOPA oxidation to dopachrome (λmax = 475 nm) by mushroom tyrosinase. 2,3-DBA and 2,5-DBA at relatively low concentration had a synergistic effect on the reaction, whereas at relatively high concentrations they inhibited the rate of DL-DOPA oxidation. The synergistic effect of 0.6-13.3 mM 2,3-DRA on the rate of DL-DOPA oxidation to dopachrome (λmax = 475 nm) was found to be due to the ability of 2,3-DBA-o-quinone (formed by the oxidation of 2,3-DBA by mushroom tyrosinase or by sodium periodate) to oxidize DL-DOPA to dopachrome (via dopaquinone) non-enzymatically. A similar explanation is likely to be valid for the synergism exerted by 2,5-DBA on the rate of DL-DOPA oxidation by mushroom tyrosinase.     

Specific removal of chlorine from the ortho-position of halogenated benzoic acids by reductive dechlorination in anaerobic enrichment cultures

Anaerobic enrichment cultures catalysing the reductive dechlorination of chlorinated benzoic acids were obtained from three fresh-water sediments collected from seven different locations. Sub-cultures from these enrichments specifically removed ortho-substituted chlorine from 2,3,6-, 2,3,5- and 2,4,6-trichlorobenzoic acid, yielding chloride and 2,5-, 3,5-, and 2,4-dichlorobenzoic acids, respectively. These reductive dehalogenations were stimulated by the addition of benzoate and/or volatile organic acids. In one of these enrichments dehalogenation of ortho- and/or para-chlorine substituents was also observed from 2,3-, 2,4-, 2,5-, and 3,4-dichlorobenzoic acid, yielding 3- and 4-chlorobenzoate. Removal of meta-chlorines was not observed in any of the enrichments.     

Degradation of polychlorinated phenols by Streptomyces rochei 303

The strain Streptomyces rochei 303 (VKM Ac-1284D) is capable of utilizing 2-chloro-,2,4-,2,6-dichloro- and 2,4,6-trichlorophenols as the sole source of carbon. Its resting cells completely dechlorinated and degraded 2-, 3-chloro-; 2,4-, 2,6-, 2,3-, 2,5-, 3,4-, 3,5-dichloro-; 2,4-, 2,6-dibromo-; 2,4,6-, 2,4,5-, 2,3,4-, 2,3,5-, 2,3,6-trichlorophenols; 2,3,5,6-tetrachloro- and pentachlorophenol. During chlorophenol degradation, a stoichiometric amount of chloride ions was released and chlorohydroquinols were formed as intermediates. In cell-free extracts of S. rochei, the activity of hydroxyquinol 1,2-dioxygenase was found. The enzyme was induced with chlorophenols. Of all so far described strains degrading polychlorophenols, S. rochei 303 utilized a wider range of chlorinated phenols as the sole sourse of carbon and energy.Abbreviations CP chlorophenol - DCP dichlorophenol - TCP trichlorophenol - TeCP tetrachlorophenol - PCP pentachlorophenol - DBrP dibromophenol - CHQ chlorohydroquinol - DCHQ dichlorohydroquinol - HHQ hydroxyhydroquinol - CHHQ chlorohydroxyhydroquinol - CC chlorocatechol - TLC thin layer chromatography - GC/MC chromato-mass-spectrometry - HPLC high-performance liquid chromatography     

Anaerobic biodegradation of chlorophenols in fresh and acclimated sludge.

We investigated the anaerobic biodegradation of mono- and dichlorophenol isomers by fresh (unacclimated) sludge and by sludge acclimated to either 2-chlorophenol, 3-chlorophenol, or 4-chlorophenol. Biodegradation was evaluated by monitoring substrate disappearance and, in selected cases, production of 14CH4 from labeled substrates. In unacclimated sludge, each of the monochlorophenol isomers was degraded. The relative rates of disappearance were in this order: ortho greater than meta greater than para. For the dichlorophenols in unacclimated sludge, reductive dechlorination of the Cl group ortho to phenolic OH was observed, and the monochlorophenol compounds released were subsequently degraded. 3,4-Dichlorophenol and 3,5-dichlorophenol were persistent. Sludge acclimated to 2-chlorophenol cross-acclimated to 4-chlorophenol but did not utilize 3-chlorophenol. This sludge also degraded 2,4-dichlorophenol. Sludge acclimated to 3-chlorophenol cross-acclimated to 4-chlorophenol but not to 2-chlorophenol. This sludge degraded 3,4- and 3,5-dichlorophenol but not 2,3- or 2,5-dichlorophenol. The specific cross-acclimation patterns observed for monochlorophenol degradation demonstrated the existence of two unique microbial activities that were in turn different from fresh sludge. The sludge acclimated to 4-chlorophenol could degrade all three monochlorophenol isomers and 2,4- and 3,4-dichlorophenol. The active microbial population in this sludge appeared to be a mixture of populations present in the 2-chlorphenol- and 3-chlorophenol-acclimated sludges, both of which could utilize 4-chlorophenol. Experiments with 14C-radiolabeled p-chlorophenol, o-chlorophenol, and 2,4-dichlorophenol demonstrated that these compounds were converted to 14CH4 and 14CO2.     

Transformation of chlorinated benzenes and toluenes by Ralstonia sp. strain PS12 tecA (tetrachlorobenzene dioxygenase) and tecB (chlorobenzene dihydrodiol dehydrogenase) gene products

The tecB gene, located downstream of tecA and encoding tetrachlorobenzene dioxygenase, in Ralstonia sp. strain PS12 was cloned into Escherichia coli DH5alpha together with the tecA gene. The identity of the tecB gene product as a chlorobenzene dihydrodiol dehydrogenase was verified by transformation into the respective catechols of chlorobenzene, the three isomeric dichlorobenzenes, as well as 1,2,3- and 1,2,4-trichlorobenzenes, all of which are transformed by TecA into the respective dihydrodihydroxy derivatives. Di- and trichlorotoluenes were either subject to TecA-mediated dioxygenation (the major or sole reaction observed for the 1,2,4-substituted 2,4-, 2,5-, and 3,4-dichlorotoluenes), resulting in the formation of the dihydrodihydroxy derivatives, or to monooxygenation of the methyl substituent (the major or sole reaction observed for 2,3-, 2,6-, and 3,5-dichloro- and 2,4,5-trichlorotoluenes), resulting in formation of the respective benzyl alcohols. All of the chlorotoluenes subject to dioxygenation by TecA were transformed, without intermediate accumulation of dihydrodihydroxy derivatives, into the respective catechols by TecAB, indicating that dehydrogenation is no bottleneck for chlorobenzene or chlorotoluene degradation. However, only those chlorotoluenes subject to a predominant dioxygenation were growth substrates for PS12, confirming that monooxygenation is an unproductive pathway in PS12.     

Influence of chlorobenzoates on the utilisation of chlorobiphenyls and chlorobenzoate mixtures by chlorobiphenyl/chlorobenzoate-mineralising hybrid bacterial strains

Chlorobenzoates (CBA) arise as intermediates during the degradation of polychlorinated biphenyls (PCBs) and some chlorinated herbicides. Since PCBs were produced as complex mixtures, a range of mono-, di-, and possibly trichloro-substituted benzoates would be formed. Chlorobenzoate degradation has been proposed to be one of the rate-limiting steps in the overall PCB-degradation process. Three hybrid bacteria constructed to have the ability to completely mineralise 2-, 3-, or 4-monochlorobiphenyl respectively, have been studied to establish the range of mono- and diCBAs that can be utilised. The three strains were able to mineralise one or more of the following CBAs: 2-, 3-, and 4-monochlorobenzoate and 3,5-dichlorobenzoate. No utilisation of 2,3-, 2,5-, 2,6-, or 3,4-diCBA was observed, and only a low concentration (0.11 mM) of 2,4-diCBA was mineralised. When the strain with the widest substrate range (Burkholderia cepacia JHR22) was simultaneously supplied with two CBAs, one that it could utilise plus one that it was unable to utilise, inhibitory effects were observed. The utilisation of 2-CBA (2.5 mM) by this strain was inhibited by 2,3-CBA (200 M) and 3,4-CBA (50 M). Although 2,5-CBA and 2,6-CBA were not utilised as carbon sources by strain JHR22, they did not inhibit 2-CBA utilisation at the concentrations studied, whereas 2,4-CBA was co-metabolised with 2-CBA. The utilisation of 2-, 3-, and 4-chlorobiphenyl by strain JHR22 was also inhibited by the presence of 2,3- or 3,4-diCBA. We conclude that the effect of the formation of toxic intermediates is an important consideration when designing remediation strategies.Abbreviations PCB Polychlorinated biphenyl - CBA Chlorobenzoate     

Effects of mono-, di- and tri-hydroxybenzoic acids on the nitrosation of propranolol: structure-activity relationship

Nitrosation of propranolol under standard conditions recommended by the World Health Organization (10mM propranolol hydrochloridre, 40mM sodium nitrite, pH 3.5) was performed in the absence and in the presence of benzoic acid and of twelve mono-, di- and tri-hydroxybenzoic acids, added to the nitrosation mixture in concentrations ranging from 2 to 40mM, in order to examine their effect on the nitrosation reaction. The yield of N-nitrosopropranol (NOP) was reduced by benzoic acid and, with potency decreasing in the following order, by 2,3,4-tri-hydroxybenzoic acid>/=3,4-tri-hydroxybenzoic acid>2,5-di-hydroxybenzoic acid>2,3-di-hydroxybenzoic acid>3-hydroxybenzoic acid>2-hydroxybenzoic acid>3,4,5-tri-hydroxybenzoic acid>4-hydroxybenzoic acid; their inhibiting effect was concentration-dependent. In contrast, 2,4-di-hydroxybenzoic acid, 2,6-di-hydroxybenzoic acid and 2,4,6-tri-hydroxybenzoic acid caused an increase in the yield of NOP that was inversely related to their concentration. 3,5-Di-hydroxybenzoic acid was substantially inactive. These findings indicate that, depending on the positions of carboxyl group and hydroxyl groups on the benzene ring, mono-, di- and tri-hydroxybenzoic acids may inhibit or hasten nitrosation reactions. As compared with benzenediols and benzenetriols [Mutat. Res. 398 (1998) 75], hydroxybenzoic acids inhibit the nitrosation of propranolol to a greater extent and have the advantage of being nonmutagenic and less toxic.     

The genotoxicity of a variety of aniline derivatives in a DNA repair test with primary cultured rat hepatocytes

The genotoxicity of a variety of aniline derivatives was examined by a DNA repair test with rat hepatocytes. Out of 37 aniline derivatives, 6 chemicals, i.e., 2,4,6-trimethylaniline (mesidine), 2,4-xylidine, 3,5-diaminobenzoic acid, 3,4-diaminochlorobenzene, 2-chloro-4-methylaniline and 4-chloro-N-methylaniline, elicited positive DNA repair responses. The results are in agreement with the bacterial mutagenicities with or without norharman of these compounds. Positive compounds of unknown carcinogenicity in the present assay, i.e., 3,5-diaminobenzoic acid, 2-chloro-4-methylaniline and 4-chloro-N-methylaniline are suspected of being potentially carcinogenic.     

Isolation and identification of two novel butenolides as products of dimethylbenzoate metabolism by Rhodococcus rhodochrous N75

Abstract 3,4-Dimethylbenzoic acid and 3,5-dimethylbenzoic acid were both oxidised by 4-methylbenzoate ( p -toluate)-grown cells of Rhodococcus rhodochrous N75 via the ortho -pathway through the intermediates 3,4- and 3,5-dimethylcatechol, respectively. Owing to the formation of the two novel dead-end metabolites, 4-carboxymethyl-2,3-dimethylbut-2-en-1,4-olide and 4-carboxymethyl-2,4-dimethylbut-2-en-1,4-olide from these substrates, 3,4- and 3,5-dimethylbenzoate did not serve as growth substrates for the strain.     

Pigment Production of Cryptococcus neoformans Grown with Extracts of Guizotia abyssinica

Brown pigment(s) formed in Cryptococcus neoformans when grown on media containing extracts of the seeds of Guizotia abyssinica cannot be extracted by common organic solvents or by 6 n HCl or 2 n NaOH. A similar pigmentation was observed in C. neoformans when grown on a medium containing caffeic acid isolated from the hydrolyzed methanol extract of G. abyssinica seeds. Its methyl ester and the diacetate thereof, as well as the following structurally related compounds, 3-hydroxytyramine, 3,4-dihydroxybenzoic acid, 3,4-dihydroxyphenylethanolamine, and 4-hydroxy-3,5-dimethoxycinnamic acid, brought about similar pigmentation. However, 2,4-, 2,5-, 2,6-, and 3,5-dihydroxybenzoic acids, tyrosine, phenylalanine, cinnamic acid, 4-hydroxycinnamic acid, and 4-hydroxy-3-methoxycinnamic acid did not cause coloration in C. neoformans.     

Transformation of Chlorinated Benzenes and Toluenes by Ralstonia sp. Strain PS12 tecA (Tetrachlorobenzene Dioxygenase) and tecB (Chlorobenzene Dihydrodiol Dehydrogenase) Gene Products

The tecB gene, located downstream of tecA and encoding tetrachlorobenzene dioxygenase, in Ralstonia sp. strain PS12 was cloned into Escherichia coli DH5α together with the tecA gene. The identity of the tecB gene product as a chlorobenzene dihydrodiol dehydrogenase was verified by transformation into the respective catechols of chlorobenzene, the three isomeric dichlorobenzenes, as well as 1,2,3- and 1,2,4-trichlorobenzenes, all of which are transformed by TecA into the respective dihydrodihydroxy derivatives. Di- and trichlorotoluenes were either subject to TecA-mediated dioxygenation (the major or sole reaction observed for the 1,2,4-substituted 2,4-, 2,5-, and 3,4-dichlorotoluenes), resulting in the formation of the dihydrodihydroxy derivatives, or to monooxygenation of the methyl substituent (the major or sole reaction observed for 2,3-, 2,6-, and 3,5-dichloro- and 2,4,5-trichlorotoluenes), resulting in formation of the respective benzyl alcohols. All of the chlorotoluenes subject to dioxygenation by TecA were transformed, without intermediate accumulation of dihydrodihydroxy derivatives, into the respective catechols by TecAB, indicating that dehydrogenation is no bottleneck for chlorobenzene or chlorotoluene degradation. However, only those chlorotoluenes subject to a predominant dioxygenation were growth substrates for PS12, confirming that monooxygenation is an unproductive pathway in PS12.     

Degradation of mono- and dichlorobenzoic acid isomers by two natural isolates of Alcaligenes denitrificans

Two strains of Alcaligenes denitrificans, designated BRI 3010 and BRI 6011, were isolated from polychlorinated biphenyl (PCB)-contaminated soil using 2,5-dichlorobenzoic acid (2,5-DCBA) and 2,4-DCBA, respectively, as sole carbon and energy sources. Both strains degraded 2-chlorobenzoic acid (2-CBA), 2,3-DCBA, and 2,5-DCBA, and were unable to degrade 2,6-DCBA. BRI 6011 alone degraded 2,4-DCBA. Growth of BRI 6011 in yeast extract and 2,6-DCBA induced pyrocatechase activity, but 2,6-DCBA was not degraded, suggesting the importance of an unsubstituted carbon six of the aromatic ring. Metabolism of the chlorinated substrates resulted in the stoichiometric release of chloride, and degradation proceeded by intradiol cleavage of the aromatic ring. Growth of both strains on 2,5-DCBA induced pyrocatechase activities with catechol and chlorocatechols as substrates. In contrast to dichlorobenzoic acids, growth on 2-CBA, benzoic acid, mono- and dihydroxybenzoic acids induced a pyrocatechase activity against catechol only. Although 2,4-DCBA was a more potent inducer of both pyrocatechase activities, its utilization by BRI 6011 was inhibited by 2,5-DCBA. Specific uptake rates using resting cells were highest with 2-CBA, except when the resting cells had been previously grown on 2,5-DCBA, in which case 2,5-DCBA was the preferred substrate. The higher rates of 2,5-DCBA uptake obtained by growth on that substrate, suggested the existence of a separately induced uptake system for 2,5-DCBA.     

Direct resolution of propranolol and bupranolol by thin-layer chromatography using cellulose derivatives as stationary phase

Cellulose triphenylcarbamate derivatives have been used as stationary phases for resolution of the enantiomers of the β-blockers propranolol and bupranolol by TLC. The derivatives examined were: cellulose trisphenylacarbamate (1), cellulose tris(2,3-dichlorophenyl carbamate) (2), cellulose tris(2,4-dichlorophenyl carbamate) (3), cellulose tris(2,6-dichlorophenyl carbamate) (4), cellulose tris (2,3-dimethylphenyl carbamate) (5), cellulose tris(3,4-dichlorophenyl carbamate) (6), cellulose tris(3,5-dichlorophenyl carbamate) (7), and cellulose tris(3,5-dimethylphenyl carbamate) (8). A variety of mobile phases were used to achieve useful separations and the effects of solvent polarity are also discussed. The best resolution of rac-propranolol was obtained on CSP 8 (R_fR = 0.26, R_fS = 0.06, α = 4.33) in mobile phase hexane:propan-2-ol (80:20 v/v). The best resolution of rac-bupranolol was obtained on CSP 5 (R_fR = 0.29, R_fS = 0.09, α = 3.22) in mobile phase hexane:propan-2-ol (80:20 v/v). These results demonstrated the potential of cellulose triphenylcarbamates as chiral stationary phases in TLC and indicate that this is potentially a useful method for the direct, simple, and rapid (within 30 min) resolution of racemates in the analytical control of enantiomeric purity. Physical aspects such as problems in cracking of the CSP, adhesion to plate, and interference of spot detection due to triphenylcarbamate chromphores are also discussed, along with the method employed to overcome them. Chirality 9:139–144, 1997. © 1997 Wiley-Liss, Inc.     

Complexes of sodium vanadate(V) with methyl alpha-D-mannopyranoside,methyl alpha- and beta-D-galactopyranoside,and selected O-methyl derivatives: a 51V and 13C NMR study

The hydroxyl group stereochemistry of complexation of sodium vanadate(V) with Me alpha-Manp, Me alpha- and beta-Galp and selected O-methyl derivatives in D(2)O was determined by 51V, 1D and 2D 13C NMR spectroscopy at pD 7.8. The 51V approach served to show the extent of complexation and the minimum number of esters formed. That of Me alpha-Manp gave rise mainly to a 51V signal at delta -515, identical with that of its 4,6-di-O-methyl derivative, which had only a 2,3-cis-diol exposed. The 13C NMR spectra contained much weaker signals of the complexes, but both glycosides showed strong C-2 and C-3 alpha-shifts of +17.3 and +10.8 ppm, respectively. As expected, Me 2,3-Me(2)-alpha-Manp, which contains a 4,6-diol, did not complex. Me Galp anomers and their derivatives showed more diversity in the structure of its oxyvanadium derivatives. Me alpha-Galp, with its 3,4-cis-diol, complexed to give rise to 51V signals at delta -495 (9%), -508 (10%), and -534 (4%). These shifts and proportions were maintained with Me beta-Galp and Me 6Me-alpha-Galp. 51V NMR spectroscopy showed that Me 3Me-beta-Galp, with its possibly available 4,6-diol, did not complex. Similarly, Me alpha-Galp+vanadate gave a 13C DEPT spectrum that did not contain an inverted signal at delta >71.4, as would be expected of a C-6 resonance suffering a strong downfield alpha-shift. Me 2,6-Me(2)-alpha-Galp, with a 3,4-cis-diol group, gave rise to two 51V signals of complexes at delta -492 (9%) and -508 (9%), showing more than one structure of oxyvanadium derivatives.     

Metabolism of and inhibition by chlorobenzoates in Pseudomonas putida P111.

Pseudomonas putida P111 was isolated by enrichment culture on 2,5-dichlorobenzoate and was also able to grow on 2-chloro-, 3-chloro-, 4-chloro-, 2,3-dichloro-, 2,4-dichloro-, and 2,3,5-trichlorobenzoates. However, 3,5-dichlorobenzoate completely inhibited growth of P111 on all ortho-substituted benzoates that were tested. When 3,5-dichlorobenzoate was added as a cosubstrate with either 3- or 4-chlorobenzoate, cell yields and chloride release were greater than those observed from growth on either monochlorobenzoate alone. Moreover, resting cells of P111 grown on 4-chlorobenzoate released chloride from 3,5-dichlorobenzoate and produced no identifiable intermediate. In contrast, resting cells grown on 2,5-dichlorobenzoate metabolized 3,5-dichlorobenzoate without release of chloride and accumulated a degradation product, which was identified as 1-carboxy-1,2-dihydroxy-3,5-dichlorocyclohexadiene on the basis of gas chromatography-mass spectrometry confirmation of its two acid-hydrolyzed products, 3,5- and 2,4-dichlorophenol. Since 3,5-dichlorocatechol was rapidly metabolized by cells grown on 2,5-dichlorobenzoate, it is apparent that 1-carboxy-1,2-dihydroxy-3,5-dichlorocyclohexadiene is not further metabolized by these cells. Moreover, induction of a functional dihyrodiol dehydrogenase would not be required for growth of P111 on other ortho-chlorobenzoates since the corresponding chlorodihydrodiols produced from a 1,2-dioxygenase attack would spontaneously decompose to the corresponding catechols. In contrast, growth on 3-chloro-, 4-chloro-, or 3,5-dichlorobenzoate requires a functional dihydrodiol dehydrogenase, yet only the two monochlorobenzoates appear to induce for it.