Biotransformation of 2,7-Dichloro- and 1,2,3,4-Tetrachlorodibenzo-p-Dioxin by Sphingomonas wittichii RW1

Aerobic biotransformation of the diaryl ethers 2,7-dichlorodibenzo-p-dioxin and 1,2,3,4-tetrachlorodibenzo-p-dioxin by the dibenzo-p-dioxin-utilizing strain Sphingomonas wittichii RW1, producing corresponding metabolites, was demonstrated for the first time. Our strain transformed 2,7-dichlorodibenzo-p-dioxin, yielding 4-chlorocatechol, and 1,2,3,4-tetrachlorodibenzo-p-dioxin, producing 3,4,5,6-tetrachlorocatechol and 2-methoxy-3,4,5,6-tetrachlorophenol; all of these compounds were unequivocally identified by mass spectrometry both before and after N,O-bis(trimethylsilyl)-trifluoroacetamide derivatization by comparison with authentic standards. Additional experiments showed that strain RW1 formed a second metabolite, 2-methoxy-3,4,5,6-tetrachlorophenol, from the original degradation product, 3,4,5,6-tetrachlorocatechol, by methylation of one of the two hydroxy substituents.     

Biotransformation of 1,2,3-tri- and 1,2,3,4,7,8-hexachlorodibenzo-p- dioxin by Sphingomonas wittichii strain RW1

Sphingomonas wittichii RW1 is able to catabolize 1,2,3,4-tetrachlorodibenzo-p-dioxin (H. B. Hong, Y. S. Chang, I. H. Nam, P. Fortnagel, and S. Schmidt, Appl. Environ. Microbiol. 68:2584-2588, 2002). Here we demonstrate the aerobic bacterial catabolism of the ubiquitous toxic diaryl ether pollutant 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin by this strain. The products of this biotransformation were identified as tetrachlorocatechol and 2-methoxy-3,4,5,6-tetrachlorophenol by comparing mass spectra recorded before and after n-butylboronate and N,O-bis(trimethylsilyl)-trifluoroacetamide derivatization with those of authentic compounds. Additional experiments showed that the less-chlorinated 1,2,3,7,8-pentachlorodibenzo-p-dioxin was not transformed by the strain RW1. The importance of substitution patterns for the degradability of individual congeners was illustrated by the fact that the 1,2,3-trichlorodibenzo-p-dioxin was catabolized to yield 3,4,5-trichlorocatechol, whereas the 2,3,7-trichlorodibenzo-p-dioxin was not attacked.     

Biodegradation of Dibenzo-p-dioxin, Dibenzofuran, and Chlorodibenzo-p-dioxins by Pseudomonas veronii PH-03

The dioxin-degrading strain Pseudomonas veronii PH-03 was isolated from contaminated soil by selective enrichment techniques. Strain PH-03 grew on dibenzo-p-dioxin and dibenzofuran as a sole carbon source. Further, 1-chlorodibenzo-p-dioxin, 2-chlorodibenzo-p-dioxin and other dioxin metabolites, salicylic acid, and catechol were also metabolized well. Resting cells of strain PH-03 transformed dibenzo-p-dioxin, dibenzofuran, 2,2',3-trihydroxybiphenyl, and some chlorodioxins to their corresponding metabolic intermediates such as catechol, salicylic acid, 2-hydroxy-(2-hydroxyphenoxy)-6-oxo-2,4-hexadienoic acid, and chlorocatechols. The formation of these metabolites was confirmed by comparison of gas chromatography-mass spectrometry (GC-MS) data with those of authentic compounds. Although we did observe the production of 3,4,5,6-tetrachlorocatechol (3,4,5,6-TECC) from 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-TCDD) with resting cell suspensions of PH-03, growth of strain PH-03 in the presence of 1,2,3,4-TCDD was poor. This result suggests that strain PH-03 is unable to utilize 3,4,5,6-TECC, even at very low concentration (0.01 mM) due to its toxicity. In cell-free extracts of DF-grown cells, 2,2',3-trihydroxybiphenyl dioxygenase, 2-hydroxy-6-oxo-6-phenyl-2,4-hexadienoic acid hydrolase, and catechol-2,3-dioxygense activities were detected. Moreover, the activities of meta-pyrocatechase and 2,2',3-trihydroxybiphenyl dioxygenase from the crude cell-free extracts were inhibited by 3-chlorocatechol. However, no inhibition was observed in intact cells when 3-chlorocatechol was formed as intermediate.     

Biotransformation of 1,2,3-Tri- and 1,2,3,4,7,8-Hexachlorodibenzo-p- Dioxin by Sphingomonas wittichii Strain RW1

Sphingomonas wittichii RW1 is able to catabolize 1,2,3,4-tetrachlorodibenzo-p-dioxin (H. B. Hong, Y. S. Chang, I. H. Nam, P. Fortnagel, and S. Schmidt, Appl. Environ. Microbiol. 68:2584-2588, 2002). Here we demonstrate the aerobic bacterial catabolism of the ubiquitous toxic diaryl ether pollutant 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin by this strain. The products of this biotransformation were identified as tetrachlorocatechol and 2-methoxy-3,4,5,6-tetrachlorophenol by comparing mass spectra recorded before and after n-butylboronate and N,O-bis(trimethylsilyl)-trifluoroacetamide derivatization with those of authentic compounds. Additional experiments showed that the less-chlorinated 1,2,3,7,8-pentachlorodibenzo-p-dioxin was not transformed by the strain RW1. The importance of substitution patterns for the degradability of individual congeners was illustrated by the fact that the 1,2,3-trichlorodibenzo-p-dioxin was catabolized to yield 3,4,5-trichlorocatechol, whereas the 2,3,7-trichlorodibenzo-p-dioxin was not attacked.     

Biostimulation and bioaugmentation to enhance dechlorination of polychlorinated dibenzo-p-dioxins in contaminated sediments

Dechlorination of spiked 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-TeCDD) was investigated in sediment microcosms from three polychlorinated dibenzo-p-dioxin and dibenzofuran (CDD/F)-contaminated sites: River Kymijoki, Finland; Gulf Island Pond, Maine; and Lake Roosevelt, Washington. Dechlorination was stimulated by addition of electron donor and halogenated priming compounds, and bioaugmentation by a mixed culture containing Dehalococcoides ethenogenes strain 195. Amendment with 1,2,3,4-tetrachlorobenzene (1,2,3,4-TeCB) promoted rapid dechlorination of 1,2,3,4-TeCDD to 2-monochlorodibenzo-p-dioxin (2MCDD) in Gulf Island Pond and River Kymijoki sediments, however, only slow dechlorination to 1,4-dichlorodibenzo-p-dioxin was observed in Lake Roosevelt sediments. The dechlorination pathway in 1,2,3,4-TeCB-amended microcosms proceeded mainly via 1,3-dichlorodibenzo-p-dioxin, with less production of 2,3-dichlorodibenzo-p-dioxin in comparison with other treatments. Microbial community analyses indicated that Dehalococcoides-like bacteria were enriched with 1,2,3,4-TeCB. Quantitative real-time PCR analysis of Dehalococcoides-specific 16S rRNA genes and the D. ethenogenes strain 195 dehalogenase gene, tceA, showed at least an order of magnitude higher gene copy numbers in the bioaugmented than in the nonbioaugmented microcosms. An active-dechlorinating population is present in the River Kymijoki and biostimulation may enhance both native Dehalococcoides spp. and the bioaugmented D. ethenogenes strain 195.     

Degradation of chlorinated biphenyl,dibenzofuran, and dibenzo-p-dioxin by marine bacteria that degrade biphenyl,carbazole, or dibenzofuran

Marine bacterial strains (BP-PH, CAR-SF, and DBF-MAK) were isolated using biphenyl, carbazole (CAR), or dibenzofuran (DF) respectively as substrates for growth. Their 16S ribosomal DNA sequences showed that the species closest to strain BP-PH, strain CAR-SF, and strain DBF-MAK are Alteromonas macleodii (96.3% identity), Neptunomonas naphthovorans (93.1% identity), and Cycloclasticus pugetii (97.3% identity), respectively. The metabolites produced suggested that strain CAR-SF degrades CAR via dioxygenation in the angular position and by the meta-cleavage pathway, and that strain DBF-MAK degrades DF via both lateral and angular dioxygenation. Polychlorinated biphenyl (KC-300) and 2,3-dichlorodibenzo-p-dioxin were partially degraded by strain BP-PH and strain DBF-MAK, while 2,7-dichlorodibenzo-p-dioxin and 2,4,8-trichlorodibenzofuran remained virtually unchanged.     

Models of 2,4,6-trinitrotoluene (TNT) initial conversion by yeasts

We devised a screening method to obtain basidiomycetous fungi capable of degrading dioxins. About 200 fungal strains were selected from more than 1500 strains by their ability to decolorize Remazol brilliant blue R dye as an indicator. To attempt to eliminate the factor of dioxin sorption by mycelia, we prepared two series of living cultures exposed either long term or short term to 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD), and compared the decreases in the levels of this chemical. In only 11 strains was there a significant difference between the two treatments. We chose Panellus stypticus strain 99-334 as a new, effective dioxin degrader, because it gave a close to 100% decrease in 2,7-DCDD levels (from an initial concentration of 10 microM) after 40 days of exposure. The detection of a metabolic intermediate (1-chloro-3,4-dihydroxybenzene) by gas chromatography-mass spectrometry analysis supported the ability of this strain to degrade 2,7-DCDD.     

Is the biotransformation of chlorinated dibenzo-p-dioxins by Sphingomonas wittichii RW1 governed by thermodynamic factors?

Density functional theory (DFT) calculations were used to explore the relationship between the biotransformation of dibenzo-p-dioxin and selected chlorinated derivatives by resting cells of Sphingomonas wittichii RW1 and measuring the thermodynamic properties of the biotransformation substrates. Sphingomonas wittichii RW1 can aerobically catabolize dibenzo-p-dioxin as well as 2,7-dichloro-, 1,2,3-trichloro-, 1,2,3,4-tetrachloro-, and 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin; however, neither the 2,3,7-trichloro- nor the 1,2,3,7,8-pentachlorodibenzo-p-dioxin was transformed to its corresponding metabolic intermediate. The experimental biotransformation rates established were apparently governed by the selected thermodynamic properties of the substrates tested.     

Phenanthrenoids from the wetland Juncus acutus

Nine 9,10-dihydrophenanthrenes, three phenanthrenes and a related pyrene have been isolated from the wetland plant Juncus acutus. The structures have been attributed by means of their spectral data and chemical correlation. 5-(1-Ethoxy-ethyl)-2-hydroxy-7-methoxy-1,8-dimethyl-9,10-dihydrophenanthrene and 5-(1-phytoxy-ethyl)-2-hydroxy-7-methoxy-1,8-dimethyl-9,10-dihydrophenanthrene, 2,7-dihydroxy-1-methyl-5-vinylphenanthrene, 2,7-dimethoxy-1,6-dimethyl-5-vinylphenanthrene and 2,7-dihydroxy-1,6-dimethylpyrene are described for the first time. Many of the compounds showed in vitro phytotoxicity against Selenastrum capricornutum, a microalga used in aquatic tests.     

Catabolism of 2,7-dichloro- and 2,4,8-trichlorodibenzofuran by Sphingomonas sp strain RW1

Due to their physicochemical and toxicological properties, polychlorinated dibenzofurans are regarded as a class of compounds providing reason for serious environmental concern. While the nonhalogenated basic structure dibenzofuran is effectively mineralized by appropriate bacterial strains, its polychlorinated derivatives are not. To elucidate the ability of the strain Sphingomonas sp RW1 to metabolize some of these chlorinated derivatives, we performed turnover experiments using 2,7-dichloro- and 2,4,8-trichlorodibenzofuran. As indicated by the oxygen-uptake rates determined for these two chlorinated dibenzofurans, Sphingomonassp RW1 can catabolize these chlorinated dibenzofurans yielding small quantities of oxidation products, which we isolated and subsequently characterized employing GC/MS and ¹H- as well as ¹³C-NMR spectroscopy. In the case of 2,7-dichlorodibenzofuran, two metabolites accumulated, which we identified as 6-chloro- and 7-chloro-2-methyl-4H-chromen-4-one. The single metabolite isolated from the turnover experiments performed with 2,4,8-trichlorodibenzofuran was unequivocally identified as 6,8-dichloro-2-methyl-4H-chromen-4-one. Received 26 April 1999/ Accepted in revised form 23 July 1999     

Degradation of 2,7-dichlorodibenzo-p-dioxin by the lignin-degrading basidiomycete Phanerochaete chrysosporium.

Under secondary metabolic conditions, the white-rot basidiomycete Phanerochaete chrysosporium degraded 2,7-dichlorodibenzo-p-dioxin (I). The pathway for the degradation of I was elucidated by the characterization of fungal metabolites and oxidation products generated by lignin peroxidase (LiP), manganese peroxidase (MnP), and crude intracellular cell-free extracts. The multistep pathway involves the degradation of I and subsequent intermediates by oxidation, reduction, and methylation reactions to yield the key intermediate 1,2,4-trihydroxybenzene (III). In the first step, the oxidative cleavage of the dioxin ring of I, catalyzed by LiP, generates 4-chloro-1,2-benzoquinone (V), 2-hydroxy-1,4-benzoquinone (VIII), and chloride. The intermediate V is then reduced to 1-chloro-3,4-dihydroxybenzene (II), and the latter is methylated to form 1-chloro-3,4-dimethoxybenzene (VI). VI in turn is oxidized by LiP to generate chloride and 2-methoxy-1,4-benzoquinone (VII), which is reduced to 2-methoxy-1,4-dihydroxybenzene (IV). IV is oxidized by either LiP or MnP to generate 4-hydroxy-1,2-benzoquinone, which is reduced to 1,2,4-trihydroxybenzene (III). The other aromatic product generated by the initial LiP-catalyzed cleavage of I is 2-hydroxy-1,4-benzoquinone (VIII). This intermediate is also generated during the LiP- or MnP-catalyzed oxidation of the intermediate chlorocatechol (II). VIII is also reduced to 1,2,4-trihydroxybenzene (III). The key intermediate III is ring cleaved by intracellular cell extracts to produce, after reduction, beta-ketoadipic acid. In this pathway, initial oxidative cleavage of both C-O-C bonds in I by LiP generates two quinone products, 4-chloro-1,2-benzoquinone (V) and 2-hydroxy-1,4-benzoquinone (VIII). The former is recycled by reduction and methylation reactions to generate an intermediate which is also a substrate for peroxidase-catalyzed oxidation, leading to the removal of a second chlorine atom. This unique pathway results in the removal of both aromatic chlorines before aromatic ring cleavage takes place.     

Anaerobic reductive dechlorination of chlorinated dioxins in estuarine sediments

The biotransformation of 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-tetraCDD) under anaerobic sulfate-reducing, methanogenic, and iron-reducing conditions was examined with anaerobic enrichment cultures established with sediment from an estuarine intertidal strait in the New York/New Jersey harbor. In addition, the effect of prior enrichment on 2-bromophenol or a mixture of 2-, 3-, and 4-bromophenol on dioxin dechlorination was examined. All enrichments were spiked with 1 ppm 1,2,3,4-tetraCDD and monitored by gas chromatography-mass spectrometry for up to a 3-year period. Reductive dechlorination was initially observed only under methanogenic conditions in the cultures enriched on all three bromophenol isomers. 1,2,3,4-TetraCDD was dechlorinated in the lateral position to 1,2,4-triCDD. The initial appearance of 1,2,4-triCDD was observed after 2 months, with further dechlorination to 1,3-diCDD within 17 months.     

Hydroxylation and Dechlorination of Tetrachlorohydroquinone by Rhodococcus sp. Strain CP-2 Cell Extracts

A cell extract of a polychlorophenol-degrading bacterium, Rhodococcus sp. strain CP-2, isolated from chlorophenol-contaminated soil, was shown to dechlorinate tetrachlorohydroquinone, the first intermediate in pentachlorophenol and 2,3,5,6-tetrachlorophenol degradation. Degradation of tetrachlorohydroquinone was catalyzed by a soluble enzyme(s). The reaction sequence for complete dechlorination involved hydroxylation and three reductive dechlorinations, producing 1,2,4-trihydroxybenzene. All chlorines were thus removed from the polychlorinated compound before ring cleavage.     

Intron-dependent accumulation of mRNA in Coriolus hirsutus of lignin peroxidase gene the product of which is involved in conversion/degradation of polychlorinated aromatic hydrocarbons

The homobasidiomycete Coriolus hirsutus coding sequences of a lignin peroxidase (LiP) gene (lip, containing six (I-VI) introns), a lip cDNA (lipc), and three lipc derivatives containing one (I), three (I-III), or five (I-V) introns were inserted into chromosome-integrating expression vector. These recombinant plasmids were introduced into C. hirustus monokaryotic strain. The transformant carrying the promoter-lipc-terminator cassette did not contain enough mRNA molecules to be detectable by Northern-blot analysis. On the other hand, all the transformants carrying cassettes of genomic lip and intron(s)-containing lipc sequences contained sufficient amounts of mRNAs to be easily detected by Northern-blot analysis. LiP activities in the culture supernatants of these transformants were found to be about five times as high as those of transformants carrying the lipc cassette (or no cassette). The culture supernatants of the transformants with high LiP activity showed remarkably high conversion activity toward pentachlorophenol (PCP) and degradation activity toward 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD). These results indicate that at least one intron (intron I) is required for accumulation of lip mRNA and its subsequent translational expression in C. hirsutus.     

Antimitotic activity and reversal of breast cancer resistance protein-mediated drug resistance by stilbenoids from Bletilla striata

Eight stilbenoids, 1-(p-hydroxybenzyl)-4,8-dimethoxyphenanthrene-2,7-diol (1), 2,7-dihydroxy-1,3-bis(p-hydroxybenzyl)-4-methoxy-9,10-dihydrophenanthrene (2), 4,7-dihydroxy-1-(p-hydroxybenzyl)-2-methoxy-9,10-dihydrophenanthrene (3), 3,3'-dihydroxy-2',6'-bis(p-hydroxybenzyl)-5-methoxybibenzyl (4), 3',5-dihydroxy-2-(p-hydroxybenzyl)-3-methoxybibenzyl (5), blestriarenes B (6) and C (7), and blestrianol A (8) have been isolated by the guidance of inhibitory effect of tubulin polymerization from the tubers of Bletilla striata (Orchidaceae). Among them, both of bisbenzyls 4 and 5 inhibited the polymerization of tubulin at IC(50) 10muM, respectively. Furthermore bisbenzyl 4 potentiated the cytotoxicity of SN-38 in BCRP-transduced K562 (K562/BCRP) cells.     

Formaldehyde-derived tetrahydroisoquinolines and tetrahydro-β-carbolines in human urine

Human urine samples were examined for the occurrence of formaldehyde-derived tetrahydroisoquinolines and tetrahydro-β-carbolines generated by condensation of the methanol oxidation product with biogenic amines. Positive results were obtained for the tryptamine condensation product 1,2,3,4-tetrahydro-β-carboline and the serotonine condensation product 6-hydroxy-1,2,3,4-tetrahydro-β-carboline as well as for the condensation products with tyramine, dopamine, adrenaline and noradrenaline 1,2,3,4-tetrahydroisoquinoline, 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, N-methyl-4,6,7-trihydroxy-1,2,3,4-tetrahydroisoquinoline, 4,6,7-trihydroxy-1,2,3,4-tetrahydroisoquinoline, and the metabolite 6-methoxy-7-hydroxy-1,2,3,4-tetrahydroisoquinoline. Negative results were obtained for N-methyl-1,2,3,4-tetrahydroisoquinoline and 6,7-di-methoxy-1,2,3,4-tetrahydroisoquinoline, N-methyl-1,2,3,4-tetrahydro-β-carboline, 6-methyl-1,2,3,4-tetrahydro-β-carboline, and 6-methoxy-1,2,3,4-tetrahydro-β-carboline in samples of chronic alcoholics as well as in the urine of healthy volunteers. No correlation between alcohol ingestion or state of alcoholization could be demonstrated.     

Experiments Directed toward the Total Synthesis of Polycyclic Terpenes

A synthetic sequence for the conversion of 6-methoxy-1,2,3,4-tetrahydronaphthalenone (I) to the bridged tetracyclic compound, 2-methoxy-6-oxo-7,8a-ethano-12-oxo-6,7,8,8a,9,10-hexahydrophenanthrene (VII) is described.

A synthetic sequence for the conversion of 2-methoxycarbonylmethyl-6-methoxy-1, 2, 3, 4-tetrahydronaphthalenone (II) to (±)-3, 16-dioxo-20-nor-hiba-5, (6)-ene is described.     

The occurrence of 2-methyl-1,2,3,4-tetrahydro-β-carboline and variation in alkaloids in Phalaris arundinacea

2-Methyl-1,2,3,4-tetrahydro-β-carboline was isolated from reed canarygrass (Phalaris arundinacea L.) and the occurrence of 2-methyl-6-methoxy-1,2,3,4-tetrahydro-β-carboline confirmed. Clones of reed canarygrass that contained N,N-dimethyltryptamine or 2-methyl-1,2,3,4-tetrahydro-β-carboline did not contain their respective methoxy or hydroxy derivatives. Five of the 12 clones tested contained either or both of 5-methoxy-N,N-dimethyltryptamine and 2-methyl-6-methoxy-1,2,3,4-tetrahydro-β-carboline. The data suggest that clones that contain gramine are not likely to contain N,N-dimethyltryptamine and/or β-carbolines. Thus, an inverse biosynthetic relationship between gramine and the tryptamine and β-carboline alkaloids seems to exist. However, further work is needed to firmly establish any such relationship between these alkaloids.     

A second [2Fe-2S] ferredoxin from Sphingomonas sp. Strain RW1 can function as an electron donor for the dioxin dioxygenase

The first step in the degradation of dibenzofuran and dibenzo-p-dioxin by Sphingomonas sp. strain RW1 is carried out by dioxin dioxygenase (DxnA1A2), a ring-dihydroxylating enzyme. An open reading frame (fdx3) that could potentially specify a new ferredoxin has been identified downstream of dxnA1A2, a two-cistron gene (J. Armengaud, B. Happe, and K. N. Timmis, J. Bacteriol. 180:3954-3966, 1998). In the present study, we report a biochemical analysis of Fdx3 produced in Escherichia coli. This third ferredoxin thus far identified in Sphingomonas sp. strain RW1 contained a putidaredoxin-type [2Fe-2S] cluster which was characterized by UV-visible absorption spectrophotometry and electron paramagnetic resonance spectroscopy. The midpoint redox potential of this ferredoxin (E'(0) = -247 +/- 10 mV versus normal hydrogen electrode at pH 8.0) is similar to that exhibited by Fdx1 (-245 mV), a homologous ferredoxin previously characterized in Sphingomonas sp. strain RW1. In in vitro assays, Fdx3 can be reduced by RedA2 (a reductase similar to class I cytochrome P-450 reductases), previously isolated from Sphingomonas sp. strain RW1. RedA2 exhibits a K(m) value of 3.2 +/- 0.3 microM for Fdx3. In vivo coexpression of fdx3 and redA2 with dxnA1A2 confirmed that Fdx3 can serve as an electron donor for the dioxin dioxygenase.     

Pyrazino[1,2-a]indoles as novel high-affinity and selective imidazoline I(2) receptor ligands

1,2,3,4-Tetrahydropyrazino[1,2-a]indoles are described as a novel class of I(2) imidazoline receptor ligands. In particular, 8-methoxy-1,2,3,4-tetrahydropyrazino[1,2-a]indole (8-OMe THPI; 3c) binds with high affinity at I(2) imidazoline receptors (K(i)=6.2 nM) and with exceptional (> or =1000-fold) selectivity relative to its affinity for I(1) imidazoline receptors, alpha(2)adrenergic receptors, and 5-HT(2A) and 5-HT(2C) serotonin receptors.