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Using scanning electrochemical microscopy (SECM) we have measured quantitatively the depletion of O2 during the photodegradation of 4-chlorophenol at supported TiO2 films for the first time and established the connection between Cl-formation and O2 depletion rates.
Nine bioactive allylphenol (anisole) derivatives (e.g. eugenol, safrole and asaricin) present in several plants of the genus Piper have been synthesized in medium to high yield via aryl cation intermediates. This expeditious metal-free procedure involves the irradiation of the corresponding chlorophenols or chloroanisoles in a polar solvent (MeCN or, better, TFE or aqueous acetonitrile) in the presence of allyltrimethylsilane. Estragole has also been synthesized starting from the corresponding fluoroderivative and diazonium salt, though in a lower yield.
5,10,15,20-Tetrakis(2,6-dichloro-3-chlorosulfophenyl)porphyrin and its tin and zinc complexes were synthesized with high yields and fully characterized. The corresponding water-soluble 5,10,15,20-tetrakis(2,6-dichloro-3-sulfophenyl)porphyrins were obtained by hydrolysis with water. An extensive photophysical study of the new water soluble porphyrinic compounds was carried out including absorption and fluorescence spectra, fluorescence quantum yields, triplet absorption spectra, triplet lifetimes, triplet and singlet oxygen quantum yields. These sensitizers were successfully used in the photodegradation of 4-chlorophenol and 2,6-dimethylphenol. A comparison is made of their efficiencies, and some mechanistic considerations are highlighted.
Peptide methionine sulphoxide reductase (MsrA; EC 1.8.4.6) is a ubiquitous enzyme catalysing the reduction of methionine sulphoxide to methionine in proteins, while the glutathione S-transferases (GSTs) are a major family of detoxification enzymes. A gene homologous to MsrA was identified in a chromosomal fragment from the bacterium Ochrobactrum anthropi, and this gene is located just downstream of a GST gene identified previously (OaGST) [Favaloro, Tamburro, Angelucci, De Luca, Melino, Di Ilio and Rotilio (1998) Biochem. J. 335, 573-579]. This raises the question of whether the products of these two genes may be involved in a common cellular protection function. To test this hypothesis, the hypothetical MsrA protein has been overexpressed in Escherichia coli as a functional 51 kDa GST fusion protein. Following cleavage with thrombin and purification, the soluble 24 kDa protein showed MsrA activity with N-acetylmethionine sulphoxide as substrate, as well as with other sulphoxide compounds. Therefore polyclonal antibodies were raised against the recombinant protein, and the modulation of MsrA in this bacterium, grown in the presence of different stimulants simulating several stress conditions, was investigated. The level of expression of MsrA was detected both by measuring the mRNA level and by immunoblotting experiments, in addition to measuring its catalytic activity. MsrA is a constitutive enzyme which is also inducible by chemical stress involving phenolic compounds such as phenol and 4-chlorophenol. Recently we reported that the GST of this bacterium, like MsrA, is only modulated by toxic chemical compounds [Favaloro, Tamburro, Trofino, Bologna, Rotilio and Heipieper (2000) Biochem. J. 346, 553-559]; therefore this is the first indication of a co-induction of the MsrA and GST enzymes during chemical stress.
Gene transfer was performed using asialo-oroso-mucoid-polylysine (ASOR-PL) conjugates to allow targeted expression of the gene in cells of hepatic origin. In a gel-electrophoretic analysis, the ASOR-PL conjugate produced a complete DNA retardation effect at the optimal ratio of 222:1 (ASOR-PL conjugate/pCMV beta-gal plasmid). The gene-transfer efficiency of the ASOR-PL conjugate was evaluated in HepG2 cells that express asialoglycoprotein receptor and NIH 3T3 cells that do not. The expression was assayed by 5-bromo-4-chloroindol-3-yl beta-D-galactopyranoside ('X-Gal') staining and Chlorophenol Red beta-D-galactopyranoside. When an expression vector for the tumour-suppressor gene p53, pCMVp53, complexed to ASOR-PL conjugate, was transfected into HepG2 cells, the exogenously provided p53 gene was detected in the HepG2 cells by PCR. To improve the efficiency of DNA delivery and expression of the therapeutic proteins poloxamer 407, a fusogenic peptide, influenza-virus haemagglutinin HA2 and chloroquine were individually incorporated into the system. The expression level of beta-galactosidase in HepG2 cells was increased by about four times by the presence of poloxamer 407, whereas the fusogenic peptide HA2 and chloroquine had no effects. When HepG2 cells were transfected with pCMVp53 in the presence of poloxamer 407, the mRNA of transfected p53 could be detected by reverse transcriptase PCR. The current findings open the possibility that a receptor-mediated gene-delivery system for hepatic gene therapy using ASOR-PL conjugate in combination with poloxamer 407 may be developed in the future.
1. A pseudomonad capable of utilizing 4-chlorophenoxyacetate (CPA) as sole source of organic carbon was isolated from soil. 2. The organism was grown in liquid culture and the following compounds were isolated and identified in culture extracts: 4-chloro-2-hydroxyphenoxyacetate, 4-chlorocatechol, beta-chloromuconate probably the cis-trans isomer and gamma-carboxymethylene-Delta(alphabeta)-butenolide. 3. Cells grown on 4-chlorophenoxyacetate were able to metabolize 4-chloro-2-hydroxyphenoxyacetate, 4-chlorocatechol and gamma-carboxymethylene-Delta(alphabeta)-butenolide without a lag period. They were not adapted to 4-chlorophenol, or to either culture isolated or synthetic beta-chloromuconate, possibly because of stereospecificity towards the cis-cis isomer. 4. On the basis of isolation and induction evidence, the following metabolic pathway is proposed for the breakdown of 4-chlorophenoxyacetate by this organism: 4-chlorophenoxyacetate --> 4-chloro-2-hydroxyphenoxyacetate --> 4-chlorocatechol --> cis-cis-beta-chloromuconate --> gamma-carboxymethylene-Delta(alphabeta)-butenolide --> maleylacetate and fumarylacetate --> fumarate and acetate.
1. Two Pseudomonas strains isolated from soil metabolized 2,4-dichlorophenoxyacetate (2,4-D) as sole carbon source in mineral salts liquid medium. 2. 2,4-Dichlorophenoxyacetate cultures of Pseudomonas I (Smith, 1954) contained 2,4-dichlorophenol, 2-chlorophenol, 3,5-dichlorocatechol and alpha-chloromuconate, the last as a major metabolite. 3. Dechlorination at the 4(p)-position of the aromatic ring must therefore take place at some stages before ring fission. 4. Pseudomonas N.C.I.B. 9340 (Gaunt, 1962) cultures metabolizing 2,4-dichlorophenoxyacetate contained 2,4-dichloro-6-hydroxyphenoxyacetate, 2,4-dichlorophenol, 3,5-dichlorocatechol and an unstable compound, probably alphagamma-dichloromuconate. 5. Cell-free extracts of the latter organism grown in 2,4-dichlorophenoxyacetate cultures contained an oxygenase that converted 3,5-dichlorocatechol into alphagamma-dichloromuconate, a chlorolactonase that in the presence of Mn(2+) ions converted the dichloromuconate into gamma-carboxymethylene-alpha-chloro-Delta(alphabeta)-butenolide, and a delactonizing enzyme that gave alpha-chloromaleylacetate from this lactone. 6. Pathways of metabolism of 2,4-dichlorophenoxyacetate are discussed.
GSTs (glutathione transferases) are a multifunctional group of enzymes, widely distributed and involved in cellular detoxification processes. In the xenobiotic-degrading bacterium Ochrobactrum anthropi, GST is overexpressed in the presence of toxic concentrations of aromatic compounds such as 4-chlorophenol and atrazine. We have determined the crystal structure of the GST from O. anthropi (OaGST) in complex with GSH. Like other bacterial GSTs, OaGST belongs to the Beta class and shows a similar binding pocket for GSH. However, in contrast with the structure of Proteus mirabilis GST, GSH is not covalently bound to Cys10, but is present in the thiolate form. In our investigation of the structural basis for GSH stabilization, we have identified a conserved network of hydrogen-bond interactions, mediated by the presence of a structural water molecule that links Ser11 to Glu198. Partial disruption of this network, by mutagenesis of Ser11 to alanine, increases the K(m) for GSH 15-fold and decreases the catalytic efficiency 4-fold, even though Ser11 is not involved in GSH binding. Thermal- and chemical-induced unfolding studies point to a global effect of the mutation on the stability of the protein and to a central role of these residues in zippering the terminal helix of the C-terminal domain to the starting helix of the N-terminal domain.
A purification procedure based on (NH4)2SO4 precipitation, and chromatography on Affi-Gel Blue, DEAE-cellulose, hydroxyapatite and Bio-Gel P-60 yields a stable 6400-fold-purified active monomeric phenol (tyrosine) sulphotransferase of 26 kDa from W10BSmL, an aplastidic mutant of Euglena gracilis var. bacillaris. The apparent Km for adenosine 3'-phosphate 5'-phosphosulphate (PAPS) is 15 microM (60 microM tyrosine as substrate); adenosine 5'-phosphosulphate is inactive. L-Tyrosine gave the lowest apparent Km (33 microM) (with PAPS at 30 microM), but tyrosine esters, tyrosinamide, L-p-hydroxyphenylglycine and a number of tyrosine dipeptides were also active, with higher Km values. Nitrophenols (m- and p-) and chlorophenols (o-, m- and p-) were active, with higher Km values than for tyrosine. D-Tyrosine was inactive as a substrate, as was D-p-hydroxyphenylglycine and a number of other tyrosine derivatives lacking the carboxy carbonyl or the amino group, or having extra ring substituents or the hydroxy group in the wrong position. Adenosine 3',5'-bisphosphate and tyrosine O4-sulphate, products of the enzyme reaction with PAPS and tyrosine as substrates, showed competitive (Ki = 20 microM) and uncompetitive (Ki = 500 microM) inhibition kinetics respectively. This appears to be the first phenol sulphotransferase to accept tyrosine as substrate. This membrane-bound enzyme may be involved in tyrosine transport as well as detoxification.
A pentachlorophenol (PCP)-degrading bacterium was isolated from possible PCP-contaminated soil from Pusan, Korea and identified as a member of the genus Pseudomonas. It used PCP as its sole source of carbon and energy. This micro-organism was capable of degrading PCP more effectively, certified by the increase in cell density and the decrease in PCP substrate. Pseudomonas sp. Bu34 was able to degrade a much higher concentration of PCP (4000 mg l-1) than any previously reported PCP-degrading bacteria and fungi and to grow in mineral salts solution containing one of a variety of chlorophenols. In non-acclimated strain Bu34, the cell number decreased from 87 to 99.9% in 75-4000 mg l-1 PCP at 24 h. In the acclimated strain the PCP toxic effect did not appear with 75 mg l-1 PCP treatment, but 1000-4000 mg l-1 PCP decreased the cell number of strain Bu34 by 25% to 24 h and then the cell number slightly increased at 48 h. Therefore, it suggested that the maximum resistance of acclimated strain Bu34 to PCP was 4000 mg l-1 PCP. We suggest that strain Bu34 could be used as a micro-organism for the bioremediation of highly PCP-contaminated soils, water or wood products.
The S(1) state geometries of benzonitrile, p-cyanophenol, o-cyanophenol, chlorobenzene, and p-chlorophenol were determined by Franck-Condon simulations and a fit of the geometry to the vibronic intensities and effective rotational constants in the harmonic limit based on ab initio force constants.
Two strains, Rhodoferax sp. P230 and Delftia (Comamonas) acidovorans MCI, have previously been shown to carry activities for the degradation of the two enantiomers of (RS)-2-(2,4-dichlorophenoxy-)propionate (dichlorprop) and (RS)-2-(4-chloro-2-methylphenoxy-)propionate (mecoprop) and, in addition, are capable of degrading phenoxyacetate derivatives 2.4-dichlorophenoxyacetate (2,4-D) and 4-chloro-2-methylphenoxyacetate (MCPA). Metabolism of the herbicides is initiated by alpha-ketoglutarate-dependent dioxygenases for both enantiomers of the phenoxypropionate herbicides and for 2,4-D. These activities were constitutively expressed for both enantiomers of dichlorprop in strain MC1 and for the Renantiomer in strain P230. Enzyme activities for the complete degradation of phenoxyacetate and phenoxypropionate herbicides were induced during incubation on either of these herbicides. Strain MC1 has about threefold higher activities for the degradation of dichlorprop and for growth on this substrate (mumax = 0.15 h(-1)) than strain P230; the maximum growth rate on 2,4-D amounts to 0.045 h(-1) with strain MC1. Dichlorprop is utilized faster than mecoprop and the R-enantiomers are cleaved with higher rates than the S-enantiomers. The degradation of the chlorophenolic intermediates seems to proceed via the modified ortho cleavage pathway as indicated by activities of the respective enzymes. The enzymatic results were supported by genetic investigations by which the presence of the genes tfdB (encoding a dichlorophenol hydroxylase), tfdC (encoding a chlorocatechol 1,2-dioxygenase) and tfdD (encoding a chloromuconate cycloisomerase) could be demonstrated in both strains by PCR after application of respective primers. The presence of the tfdA gene (encoding a 2,4-D/alpha-ketoglutarate dioxygenase) was only shown for strain P230 but was lacking in strain MC1. Sequence analysis of the tfd gene fragments revealed high homology to the degradative genes of other proteobacterial strains degrading chloroaromatic compounds. Strain MC1 carries a plasmid of about 120 kb which apparently harbors herbicide degradative genes as concluded from deletion mutants which have lost 2,4-D[phenoxalkanoate]/alpha-ketoglutarate dioxygenase activities for cleavage of the R- and S-enantiomer, and of 2,4-D. For strain P230, no plasmid could be demonstrated; the activity was stably conserved in this strain during growth under nonselective conditions.
The German Environmental Survey (GerES) is a cross-sectional probability study to determine the exposure of the general population to environmental contaminants. The study was repeated for the third time in 1998 (GerES III). Again, a stratified random procedure was used to select the study population taking into account the parameters gender, age, community size and place of residence (West- or East-Germany). A total of 4822 persons between 18 and 69 years of age from 120 localities participated in GerES III. Human biomonitoring comprised the determination of arsenic, cadmium, mercury, metabolites of polycyclic aromatic hydrocarbons (PAH), pentachlorophenol (PCP), other chlorophenols, precious metals (gold, platinum, iridium), nicotine, and cotinine in urine.
1. The biotransformation of a single i.p. dose of [14C]2-chloroaniline (1.0 mmol/kg, approximately 60 microCi/rat) was investigated in the urine and faeces of the male Fischer 344 rat. 2. During 24 h, 53.1% of the administered radioactivity was eliminated into the urine, while < 1% of the radioactivity appeared in the faeces. 3. The major biotransformation pathways were para-hydroxylation and sulphate conjugation. 4-Amino-3-chlorophenyl sulphate was the major urinary metabolite comprising 31.6% of total urinary radioactivity. The para-hydroxylated metabolite, 4-amino-3-chlorophenol (10.8%), and its O-glucuronide conjugate (3.7%) were also urinary metabolites. The formation of direct conjugates of 2-chloroaniline, the N-sulphate and N-glucuronide, was significant with as much as 18.6 and 8.6%, respectively, of these metabolites excreted in the urine. The parent compound, 2-chloroaniline, accounted for 16.9% of urinary radioactivity. 4. N-Acetylated products were minor metabolites present in urine as 2-chloro-4-hydroxyacetanilide and its sulphate or glucuronide conjugate. Neither 2-chloroacetanilide nor its oxidation products, 2-chloroglycolanilide and 2-chlorooxanilic acid, were urinary metabolites.
Studies were carried out to investigate possible contamination of pheasants with residues of lindane used as seed dressings for the control of wireworms in cereal crops. One group of laying hen pheasants was fed 20 mg of lindane-14C in gelatin capsules while another group was fed wheat seed treated with 100 ppm of lindane-14C for 15 days. Residues in eggs were monitored for about 70 days. Residues in chicks hatched from the eggs were also measured. Concentrations of residues in muscle, liver, brain, and fatty tissues were determined at various times. 14C-labeled residues in eggs increased sharply in the capsule treated group and reached an average maximum of about 19 ppm in 8 days. This level decreased to less than 0.5 ppm in about 50 days. Residues in eggs from the pheasants fed treated seed increased gradually to an average maximum of 17 ppm 22 days after commencement of the feeding program. This level decreased to less than 0.5 ppm 50 days later. Residues in hatched chicks were significantly lower than those in the eggs. Highest concentrations of residues were found in fatty tissues which decreased to non-detectable level in 6 months. Several chlorobenzene metabolites were identified in egg yolk and chick tissues. Chlorophenolic metabolites were indicated only in chicks.
Everted sacs of rat small intestine metabolized crufomate (4-tert-butyl-2-chlorophenyl methyl methylphosphoramidate) under in vitro conditions to form six 14C-labeled metabolites in quantities sufficient for isolation and identification. These metabolites were 4-tert-butyl-2-chlorophenyl methyl phosphoramidate (25%), 2-chloro-4(2-hydroxy-1,1-dimethylethyl)phenyl methyl methylphosphoramidate (19%), 2-[3-chloro-4-[[(methoxy) (methyl-amino)phosphoinyl]oxy]phenyl]-2-methylpropionic acid (2%), 4-tert-butyl-2-chlorophenol (0.8%) and its glucuronide (6%), and the aromatic glucuronide of 2-chloro-4(2-hydroxy-1,1-dimethylethyl)phenol (1%). These intestinal metabolites may represent precursory stages in the overall metabolism of crufomate.
The fate of pentachlorophenol (PCP) in soil under natural conditions was investigated. It was revealed that the total amount of PCP significantly decreased when soil was inoculated by Streptomyces rochei 303, a strain-destructor of chlorophenols. The products of PCP transformation, such as tetra- and trichlorophenols, pentachlorobenzene, chlorinated dioxins, were identified after the first month of the experiment. Their quantity was less in the variant with the introduced strain compared to control.
Cement based stabilization/solidification systems have been widely used to minimize leaching of contaminants from hazardous wastes, particularly for stabilization of wastes that contain heavy metals or other inorganic materials. However, few researchers have demonstrated that stabilization/solidification systems can adequately trap and retain organics present in these wastes. Identification of additives that can be used with the inorganic binder to retain organics has attracted much interest. Activated carbon has been found to be the best additive for immobilizing organic contaminants, but it is generally too expensive for routine use. The use of powdered reactivated carbon was studied in this research to prevent organics from leaching from solidified/stabilized waste forms because it is effective and more economically competitive. The Shrinking Unreacted Core leach test method was employed in this study. Stabilization/solidification waste forms were prepared using five organic compounds--phenol, 2-chlorophenol, chlorobenzene, aniline and Methyl Ethyl Ketone (MEK)--that were spiked into the waste forms at two concentration levels each. Reactivated carbon amounts of 0% (as control), 1% and 2% (w/w) were added into each waste form batch to determine how effectively the reactivated carbon can immobilize those organics and the optimum dose of reactivated carbon addition required. Leachate samples were analyzed using gas chromatography. Results show that 1% reactivated carbon addition is generally enough to reduce leaching of all of the organic contaminants by more than 70%, while a 2% addition reduces leaching for most of the organics to less than 1%.