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Degradation of benzene and other aromatic hydrocarbons by anaerobic bacteria
Weelink, S.A.B. - \ 2008
Wageningen University. Promotor(en): Fons Stams. - S.l. : S.n. - ISBN 9789085852391 - 128
benzeen - aromatische koolwaterstoffen - microbiële afbraak - benzene - aromatic hydrocarbons - microbial degradation
Accidental spills, industrial discharges and gasoline leakage from underground storage tanks have resulted in serious pollution of the environment with monoaromatic hydrocarbons, such as benzene, toluene, ethylbenzene and xylene (so-called BTEX). High concentrations of BTEX have been detected in soils, sediments and groundwater. The mobility and toxicity of the BTEX compounds are of major concern. In situ bioremediation of BTEX by using naturally occurring microorganisms or introduced microorganisms is a very attractive option. BTEX compounds are known to be transformed (or degraded) by microorganisms under aerobic and anaerobic conditions. As BTEX compounds are often present in the anaerobic zones of the environment, anaerobic bioremediation is an attractive remediation technique. The bottleneck in the application of anaerobic techniques is the lack of knowledge about the anaerobic biodegradation of benzene. In particular, little is known about the bacteria involved in anaerobic benzene degradation and the anaerobic benzene degradation pathway has still not been elucidated. The aim of the research presented in this thesis was to gain more insight in the degradation of benzene and other aromatic hydrocarbons by anaerobic bacteria. In particular, the physiology and phylogeny of the bacteria responsible for the degradation were studied and the results have been presented in this thesis.
Anaerobic benzene and toluene degradation was studied with different electron acceptors in batch experiments inoculated with material from an aquifer polluted by BTEX-containing landfill leachate (Banisveld landfill near Boxtel, The Netherlands). Benzene was not degraded during one year of incubation. Toluene degradation, on the other hand, was observed with nitrate, MnO2 and Fe(III)NTA as electron acceptors. After further enrichment and several isolation attempts, a novel betaproteobacterial bacterium, strain G5G6, was obtained in pure culture. Strain G5G6 is able to grow with toluene as the sole electron donor and carbon source, and amorphous and soluble Fe(III)-species, nitrate and MnO2 as electron acceptors. Strain G5G6 has several other interesting physiological and phylogenetic characteristics, which will be subject of future research. Strain G5G6 represents a novel species in a novel genus for which we propose the name Georgfuchsia toluolica.
In general, aerobic degradation of BTEX is a faster process than anaerobic BTEX degradation. However, for a number of reasons application of oxygen-dependent processes in the subsurface are technically and financially often not appealing. Therefore, an alternative bioremediation strategy would be to introduce oxygen in an alternative way, e.g. by in situ production. Chlorate reduction is a way to produce molecular oxygen in situ under anaerobic conditions. The formation of oxygen during chlorate reduction may result in rapid oxidation of compounds which are slowly degraded under anaerobic conditions; an example of such a compound is benzene. Therefore, benzene degradation coupled to the reduction of chlorate (ClO3-) was studied in this thesis. With mixed material from a wastewater treatment plant and soil samples obtained from a location contaminated with benzene, a benzene-degrading chlorate-reducing stable enrichment culture was obtained. This stable enrichment consisted of about five different bacterial species. Cross feeding involving interspecies oxygen transfer is a likely mechanism in this enrichment. One of these species, strain BC, was obtained in pure culture. Phylogenetic analysis showed that strain BC is a Alicycliphilus denitrificans strain. Strain BC is able to degrade benzene in conjunction with chlorate reduction. Oxygenase genes putatively encoding the enzymes performing the initial steps in aerobic degradation of benzene, were detected in strain BC. This demonstrates the existence of aerobic benzene bacterial biodegradation pathways under essentially anaerobic conditions. Thus, aerobic pathways can be employed under conditions where no external oxygen is supplied.
The new insights into toluene degradation under anaerobic conditions and benzene degradation coupled to chlorate reduction, as described in this thesis, can be applied for the improvement or development of in situ bioremediation strategies for BTEX contamination.
Toxicogenomics: Applications of new functional genomics technologies in toxicology
Heijne, W.H.M. - \ 2004
Wageningen University. Promotor(en): Peter van Bladeren; John Groten, co-promotor(en): B. van Ommen. - [S.I.] : S.n. - ISBN 908504121X - 213
toxicologie - genexpressie - trichloorethyleen - benzeen - toxicogenomica - toxicology - gene expression - trichloroethylene - benzene - toxicogenomics
Growth of fungi on volatile aromatic hydrocarbons: environmental technology perspectives
Prenafeta Boldú, F.X. - \ 2002
Wageningen University. Promotor(en): W.H. Rulkens; Tim Grotenhuis; J.W. van Groenestijn. - S.l. : S.n. - ISBN 9789058087478 - 115
schimmels - bodemschimmels - metabolisme - benzeen - tolueen - xyleen - biodegradatie - fungi - soil fungi - metabolism - benzene - toluene - xylene - biodegradation
The present study aimed the better understanding of the catabolism of monoaromatic hydrocarbons by fungi. This knowledge can be used to enhance the biodegradation of BTEX pollutants. Fungi with the capacity of using toluene as the sole source of carbon and energy were isolated by enriching environmental polluted soil and groundwater samples in solid state-like batches, air biofilters, and liquid cultures. Incubation conditions in the latter combined acidic pH and low water activity. The isolates were identified as <em>Cladophialophora</em> sp. (two strains), <em>Exophiala</em> sp., <em>Leptodontium</em> sp. and <em>Pseudeurotium zonatum</em> . The previously isolated toluene-growing fungus <em>Cladosporium sphaerospermum</em> was also included in the present study. The genera <em>Cladophialophora</em> , <em>Exophiala</em> (both anamorphs of <em>Capronia</em> ), and <em>Cladosporium</em> are classified into the black yeast-like and allied fungi. Members in this group are characterized for being cosmopolitan saprobes and opportunistic human pathogens. Growth curves showed that fungi grew on toluene with biomass doubling times of about 2 to 3 days. Some of the strains also grew on ethylbenzene and styrene. Kinetics of toluene degradation by cell suspensions followed the Michaelis-Menten model. Depending upon strain, the apparent <em>K</em><sub>m</sub></font>SIZE=2>for toluene oxidation ranged from 5 to 22 μM. The toxicity of toluene, measured as the IC50 value, ranged from 2.4 to 4.7 mM. The metabolic pathway for the oxidation of toluene was studied in a variety of fungi by using fluorinated toluene analog</font>SIZE=2>ues and by determining the formed metabolites by <sup>19</SUP>F NMR. Oxidation was similar in all toluene-assimilating fungi in which the initial conversion took place at the alkyl group. Toluene was initially oxidized to benzyl alcohol and benzoate, which was further hydroxylated at the ring first to 4-hydroxy-benzoate, and then to catecholic compounds. The latter served as substrates for the opening of the ring and further metabolism through the 3-oxoadipate pathway. The lack of significant benzene biodegradation by these fungi might be related with the incapacity of performing the ring hydroxylation. Oxidation of toluene at the aromatic ring was demonstrated for the zygomycete <em>Cunninghamella echinulata</em> . However, conversion rates here were very low in comparison with the assimilative metabolism and it occurred only co-metabolically.</p><p>The substrates interactions during the degradation of BTEX mixtures were studied with the isolate <em>Cladophialophora</em> sp. strain T1. This fungus grew on a model water-soluble fraction of gasoline that contained all six BTEX components. Benzene was not metabolized but the alkylated benzenes (TEX) were degraded by a combination of assimilation and co-metabolism. Toluene and ethylbenzene were used as sources of carbon and energy whereas <em>ortho</em> - and <em>meta</em> -xylene were co-metabolized to phthalates as end-metabolites. <em>Para-</em> xylene was not degraded in complex BTEX mixtures but, in combination with toluene, it appeared to be mineralized. The metabolic profiles and the inhibitory nature of the substrate interactions indicated that TEX were degraded at the side-chain by the same monooxygenase enzyme. The growth of the strain T1 was also studied in sterile and non-sterile soil microcosms contaminated with a mixture of BTEX and MTBE. Inoculation with the fungus increased the degradation rates in soil after long exposure to BTEX and a low soil pH. Comparison of the biodegradation rates measured in presence and absence of indigenous bacteria, and the fungal inoculum suggested that the main interaction between indigenous and inoculated BTEX-degrading microorganisms was commensalistic. Alkylbenzenes were all degraded by the fungus, but benzene degradation required the activity of the indigenous soil microflora. MTBE could not be biodegraded. The presence and identity of the fungal inoculum in soil was confirmed at the end of the experiments by PCR-TGGE analysis of SSU of fungal 18S rDNA.</p><p>Fungi utilizing aromatic hydrocarbons can advantageously be used for biodegradation of BTEX in air biofilters and in soil. Fungal biodegradation is similar in kinetic terms to that of bacteria and posses a higher tolerance to adverse environments. However, factors limiting the application of fungi concern the lack of benzene degradation and the potential pathogenicity to humans.
Long-term bioconcentration kinetics of hydrophobic chemicals in Selenastrum capricornutum and Microcystis aeruginosa
Koelmans, A.A. ; Woude, H. van der; Hattink, J. ; Niesten, D.J.M. - \ 1999
Environmental Toxicology and Chemistry 18 (1999). - ISSN 0730-7268 - p. 1164 - 1172.
benzeen - chloride - polychloorbifenylen - hydrofobiciteit - waterverontreiniging - microcystis aeruginosa - algen - cyanobacteriën - ecotoxicologie - bioaccumulatie - aquatische ecosystemen - benzene - polychlorinated biphenyls - hydrophobicity - water pollution - algae - cyanobacteria - ecotoxicology - bioaccumulation - aquatic ecosystems
Biotransformation of toluene, benzene and naphthalene under anaerobic conditions
Langenhoff, A.A.M. - \ 1997
Agricultural University. Promotor(en): A.J.B. Zehnder; G. Schraa. - S.l. : Langenhoff - ISBN 9789054856603 - 131
microbiële afbraak - benzeen - derivaten - microbial degradation - benzene - derivatives
<p>Aromatic hydrocarbons are widespread in nature, due to increasing industrial activity, and often contribute to polluted soils, sediments, and groundwater. Most of these compounds are toxic at relatively high concentrations, but some are already carcinogenic at very low concentrations, e.g. benzene. A growing awareness of the health risks associated with contamination has directed research to the removal or degradation of such compounds. The use of microorganisms to degrade toxic compounds (bioremediation) is a relatively slow process compared to traditional, chemical methods, but it is a natural process, mostly very specific and low in costs. A review of the available information on the microbial degradation of aromatic compounds is given in chapter 1. The anaerobic degradation is emphasized, since in many polluted environments oxygen is limiting and anaerobic processes will prevail. In the absence of oxygen, compounds like nitrate, metalions (Fe <sup>3+</SUP>and Mn <sup>4+</SUP>), sulfate, and carbondioxide, have taken over the function of oxygen as a terminal electron acceptor. In addition, the first transformation reactions differ from those in aerobic processes. Oxygenases are no longer ftinctioning and the degradation of oxygenated aromatic compounds, like benzoate and phenol, is known to occur via e.g. reduction, dehydroxylation and dehydrogenation of the aromatic ring. Information on the anaerobic degradation of mono- and polycyclic aromatic hydrocarbons without functional groups, like toluene, benzene, and naphthalene, is scarse. To gain more insight in the possibilities and limitations of the anaerobic degradation of these aromatic compounds, their behaviour in anaerobic sediment columns was followed. Toluene, benzene, and naphthalene were chosen as model compounds under methanogenic, sulfate-, iron-, manganese-, and nitrate-reducing conditions (Chapter 2). Toluene was transformed readily (within 1 to 2 months), while benzene was recalcitrant over the test period of 375-525 days under all redox conditions tested. Naphthalene was partly transformed in the column with nitrate or manganese as electron acceptor present; the addition of benzoate had a positive effect on the degradation of naphthalene in the column with nitrate. In the column with sulfate, the majority of the added naphthalene disappeared. No effect on the degradation of naphthalene was observed after adding and omitting an easier degradable substrate. [ <sup>14</SUP>C]naphthalene was used to confirm the disappearance to be the result of degradation; two third of the naphthalene was converted to CO <sub>2</sub> .<p>Numerous attempts have been made for further enrichment of sulfatereducing, naphthalene degrading bacteria (Chapter 3). Unfortunately, the observed degradation of naphthalene in a sediment column could not be obtained in batch cultures, despite the large variety of tested enrichment conditions (different naphthalene concentrations, inoculum. size, medium composition, extra additions etc.). A toxic effect of naphthalene on sulfate- reducing bacteria could not be found.<p>Toluene degradation in the columns was demonstrated under all redox conditions tested. Chapter 4 describes the degradation of toluene in freshly started sediment columns, to which either amorphous or highly crystalline manganese oxide had been added. In batch experiments with material from these columns as inoculum, the degradation of toluene to C0 <sub>2</sub> and the formation of biomass under manganese-reducing conditions was demonstrated. The oxidation of toluene was found to be coupled to the reduction of Mn(IV), and the rate of oxidation was found to be lower with the crystalline than with the amorphous manganese oxide. Upon successive transfers of the enrichment cultures, the toluene degrading activity would decrease in time. The activity could only be maintained in the presence of sterilized Rhine river sediment or its supernatant. Without the sediment, but in the presence of solids like teflon beads, glass beads, bentonite, vermiculite and sterilized granular sludge, the toluene degrading activity completely disappeared after 4 to 5 transfers. Furthermore, a direct contact between the bacteria and the manganese oxide was found to be advantageous for a rapid toluene degradation. The degradation rate could further be increased by adding organic ligands such as oxalic acid or nitrilotriacetic acid (NTA).<p>The highly purified enrichment culture LET-13, which degrades toluene with manganese oxide as electron acceptor, was obtained via repeated dilution series, and is described and characterized in chapter 5. LET-13 was able to degrade a variety of substituted monoaromatic compounds like (p-hydroxy) benzylalcohol, (p-hydroxy) benzaldehyde, (p-hydroxy) benzoate, cresol, and phenol. Benzene, ethylbenzene, xylene and naphthalene were not degraded under the experimental conditions used. The degradation of toluene occurred via hydroxylation of the methyl group to benzoate, and a possible side reaction can lead to the formation of cresol.<p>All organisms in the culture look similar; motile rods which are gram negative, oxidase negative and catalase negative. The culture was partly identified with phylogenetic analysis of cloned rDNA sequences. The phylogenetic analysis showed that at least two major groups of bacteria are present. One group of bacteria belongs to the <em>Bacteroides- Cytophaga</em> group, and one group consists of members of the β-subclass of the <em>Proteobacteria.</em><p>Finally, the results from this research are discussed in relation to their relevance for soil bioremediation technologies.
|Transformation of low concentrations of 3-chlorobenzoate by Pseudomonas sp. strain B13: kinetics and residual concentrations.
Tros, M.E. ; Schraa, G. ; Zehnder, A.J.B. - \ 1996
Applied and Environmental Microbiology 62 (1996). - ISSN 0099-2240 - p. 437 - 442.
benzeen - chloride - derivaten - hexachloorbenzeen - microbiële afbraak - pseudomonas - benzene - derivatives - hexachlorobenzene - microbial degradation
Quantitative structure activity relationships for the biotransformation and toxicity of halogenated benzene - derivatives : implications for enzyme catalysis and reaction mechanisms
Cnubben, N.H.P. - \ 1996
Agricultural University. Promotor(en): C. Veeger; Ivonne Rietjens. - S.l. : Cnubben - ISBN 9789054855200 - 272
biotransformatie - benzeen - derivaten - cytochroom p-450 - structuuractiviteitsrelaties - reactiemechanisme - biotransformation - benzene - derivatives - cytochrome p-450 - structure activity relationships - reaction mechanism - cum laude
<br/>Organisms are frequently exposed to low molecular weight xenobiotic compounds. An advanced enzymatic machinery modifies these compounds into more hydrophilic metabolites which are subsequently excreted from the body. This process of biotransformation aims to detoxify bodyforeign compounds. Ironically, reactive intermediates may also be formed during the biotransformation process and can interact with macromolecules or receptors, with possible toxicological consequences (bioactivation). Toxicological testing of all new and existing compounds is a moneyand time-consuming problem and therefore alternatives are urgently needed. The main objective of the studies decribed in this thesis, which is outlined in <strong>chapter 1</strong> , was to describe QSARs (quantitative structure-activity relationships) for the biotransformation and toxicity of halogenated aminoand nitrobenzene derivatives, Special attention was focussed on the most important phase I biotransformation enzyme involved, the cytochrome P450 system. In addition, in the course of the investigations some attention was paid to the glutathione/glutathione S-transferase dependent phase 2 biotransformation pathway.<p><strong>Chapter 2</strong> gives an overview of the biotransformation enzymes primarily involved in the metabolism of halogenated amino- and nitrobenzene derivatives with respect to their function, regulation, occurrence, molecular/biochemical mechanisms and role in bioactivation and detoxication of xenobiotics. Biotransformation of amino- and nitrobenzene derivatives plays a crucial role in the generation of reactive intermediates assumed to contribute to the toxicity of this class of compounds. Since biotransformation of nitro- and aminobenzene derivatives is known to include pathways leading to both detoxication and bioactivation, insight into factors that direct the rates and regioselectivities of biotransformation processes or interactions with nucleophiles, like macromolecules or the tripeptide glutathione, will help to gain insight in factors that direct processes of, and chances on, bioactivation or detoxication of these compounds. Regioselective enzymatic conversion of relatively small aromatic substrates in the relatively large and aspecific active sites of cytochromes P450 is assumed to depend predominantly on chemical characteristics of the substrates, whereas for relatively large substrates stereoselective positioning through steric constraints of the protein core in the active site becomes more important (see also <strong>chapter 2</strong> ). Therefore, the relative differences in reactivity of various sites in a small aromatic compound can be expected to affect the possibilities for enzymatic conversion of the molecule. These considerations prompted us to investigate whether computer calculated molecular orbital characteristics of halogenated benzene derivatives, in combination with insight into the molecular mechanisms of their enzymatic conversion, could provide a basis for the prediction of their metabolic fate.<p><strong>Chapter 3</strong> presents a study on the regioselectivity and underlying mechanisms for the cytochrome P450-catalyzed aromatic hydroxylation of monofluoroanilines. This study provides insight into the molecular mechanism of the cytochrome P450-catalyzed aromatic hydroxylation of molecules containing a heteroatom as well as factors that influence the regioselectivity of hydroxylation. Three mechanisms for aromatic hydroxylation can be proposed; hydrogen abstraction (I), or electron abstraction followed by proton release (II) both leading to formation of a NH <strong><sup><font size="+1">.</font></SUP></strong> radical. Upon rearrangement of the radical and OH- rebound from the (FeOH) <sup><font size="-2">3+</font></SUP>species, the aminophenol product is formed. Aromatic hydroxylation might also proceed by a direct interaction of the high-valent iron-oxo cytochrome (FeO) <sup><font size="-2">3+</font></SUP>intermediate with the π-electrons of the aromatic ring resulting in a so-called σ-adduct (III), which rearranges to the aminophenol, either directly or through formation of epoxides and/or ketones as intermediates. First, it was demonstrated that the regioselectivity of the aromatic hydroxylation was influenced by the position of fluoro-substituents at the aniline-ring and that the observed regioselectivity for hydroxylation of these small aromatics was not influenced by the relatively aspecific and large hydrophobic active sites of the cytochromes P450. As expected, a fluorine-substituent induces an effect on the electronic characteristics of the aniline-molecule and this effect was quantified using molecular orbital calculations. Considering the possible molecular mechanisms for aromatic hydroxylation, it appeared that the observed <em>in vitro</em> and <em>in vivo</em> regioselectivity correlated best with the frontier orbitals of importance for a direct interaction of the (FeO) <sup><font size="-2">3+</font></SUP>species with the π-electrons of the aromatic molecule <em>e g.</em> the density distribution of the HOMO/HOMO-1. The spin density distribution of the NH <strong><sup><font size="+1">.</font></SUP></strong> radicals -a parameter of importance for the hydrogen abstraction as well as electron abstraction plus proton release mechanisms- could not explain the observed regioselectivities, indicating that the regarding mechanisms are less likely. In a later study it was demonstrated that for a series of fluorobenzenes the regioselectivity for aromatic hydroxylation could even be predicted on the basis of the frontier orbital density distribution for electrophilic attack within 6% accuracy (r=0.96) [Rietjens <em>et al.,</em> 1993]. For the fluoroanilines the C4 position is hydroxylated to higher extent, whereas the C2/C6 positions are hydroxylated to an extent lower than their chemical reactivity predicts. This deviation might result from steric hindrance of the amino moiety for electrophilic attack, a stereoselective positioning of the substrate through an interaction of the amino moiety with amino acid residues in the active site as has been described for P450 debrisoquine 4-hydroxylase, or a dipole-dipole or electronic interaction between the substrate and the activated cytochrome P450 (FeO) <sup><font size="-2">3+</font></SUP>species. It is stressed here that the juxtaposition of these small substrates in the active site with respect to the Fe <sup><font size="-2">3+</font></SUP>resting state as determined on the basis of <sup><font size="-2">1</font></SUP>H-NMR T1 relaxation or cristallography studies, might not represent the actual orientation with respect to the reactive (FeO) <sup><font size="-2">3+</font></SUP>intermediate actually performing the hydroxylation step [Koerts <em>et al.,</em> 1995].<p>In <strong>chapter 4</strong> the aromatic hydroxylation of anilines was further investigated with special emphasis on possible relationships between kinetic parameters and both the physicochemical and electronic substrate characteristics. This was done in order to provide a basis for molecular orbital based quantitative structure activity relationships (MO-QSARs) for kinetic characteristics of the cytochrome P450-mediated aromatic hydroxylation of a homologous series of aniline-derivatives. It was demonstrated that the k <sub><font size="-1">cat</font></sub> for C4-hydroxylation in a series of substituted anilines strongly correlates with the HOMO energy of the anilines for the iodosobenzene supported P450 reaction in isosafrole induced microsomes. This observation is in accordance with a mechanism that proceeds by an initial electrophilic interaction of the (FeO) <sup><font size="-2">3+</font></SUP>intermediate with the frontier ic electron of the aniline-substrate. In a NADPH/oxygen-supported P450 system, however, it was demonstrated that the interaction of the (FeO) <sup><font size="-2">3+</font></SUP>species on the aniline-substrate is no longer rate-limiting, and therefore cannot be described by this QSAR. However, when the electrophilic reactivity of the substrates becomes too low, as is the case for 2,3,5,6-tetrafluoroaniline (E <sub><font size="-1">HOMO</font></sub> of -9.24 eV) the initial attack of the cytochrome (FeO) <sup><font size="-2">3+</font></SUP>on the substrate might become the rate-limiting step in the overall catalysis. The relatively low conversion rate observed for aromatic hydroxylation of fluorobenzenes for example [Rietjens <em>et al.,</em> 1993], might be explained by the relatively low electrophilic reactivity of these compounds. If the resulting metabolite is less toxic than its parent compound, a decreased conversion might have implications for the toxicity and the other way around.<p>To fully describe the cytochrome P450-mediated biotransformation of these aniline- derivatives, a sensitive and efficient analytical technique was developed for the detection and quantification of 2-aminophenols ( <strong>chapter 5</strong> ). The principle of the method was based on a dimerization reaction of 2-aminophenols to an intensively colored 2- hydroxy-isophenoxazin-3-one in an acidic environment using ferric ions as the catalyst. It was demonstrated that this method was also applicable for the detection and quantification of halogenated 2-aminophenol derivatives.<p>Besides aromatic hydroxylation reactions, also oxidative dehalogenation reactions mediated by cytochromes P450 were investigated. Halogen substituents are often introduced into molecules to block positions at the aromatic ring of drugs or agrochemicals for bioactivation or biodegradation. Fluorine substituents are frequently used for this purpose, due to the strong C-F bond, and a Van der Waals radius that almost equals that of a hydrogen. In order to study the molecular mechanism of oxidative aromatic dehalogenation as well as the consequences of halogen substitution for regioselective hydroxylation and the formation of reactive intermediates, the study described in <strong>chapter 6</strong> was undertaken. Using halogenated anilines as the model compound, the effect of a varying halogen substituent patterns on the cytochrome P450-catalyzed dehalogenation of 4- halogenated anilines to 4-aminophenols was investigated. In the case of C4-fluorinated aniline derivatives, the cytochrome P450-mediated metabolic pathway has been unequivocally demonstrated to result in direct formation of a reactive 1,4- benzoquinoneimine and fluoride anions the primary reaction products [Rietjens et al., 1993]. These reactive benzoquinoneimine metabolites may interact with cellular macromolecules and hence can lead to destruction of molecules essential to living cells.<p>The study described in <strong>chapter 6</strong> clearly demonstrated that upon the cytochrome P450-mediated oxidative dehalogenation to the primary reactive 1,4-benzoquinoneimine a fluorine substituent at C4 position of the aromatic aniline-ring was more easily eliminated than a chloro-, bromo- or iodo-substituent. A similar decrease in dehalogenation was observed in a NADPH/0 <sub><font size="-2">2</font></sub> supported microsomal P450 system, as well as in a tBuOOH supported microsomal P450 system, or a system with purified reconstituted P4502B1, or in a system with the heme-based mini-enzyme microperoxidase 8. This indicates that the decrease in dehalogenation was not a result of a change in rate-limiting steps in the P450 catalysis or a change in the contribution of P450 enzymes with a change in the halogen substituent. Structureactivity relationship principles were applied to investigate the reaction mechanism of dehalogenation. The results obtained strongly indicate that the possibilities for the cytochrome P450-mediated dehalogenation of 4-halogenated anilines to 4-aminophenol metabolites are dependent on i) the characteristics of the halogen that has to be eliminated, the most electronegative and smallest halogen (fluorine) being the one most easily eliminated and ii) the electron-withdrawing capacities of the other substituents in the aromatic ring, electron-withdrawing substituents decreasing the relative rate of the reaction. The conclusion that not only a fluoro-, but also a chloro-, bromo- and iodo- substituent, is eliminated as a halogen anion serves as the best explanation for the observations in this study. In addition, it was demonstrated that blocking the C4-position for aromatic hydroxylation by cytochromes P450 resulted in a metabolic switch from dehalogenation and 4-aminophenol formation, to formation of 2-aminophenol- and N- hydroxyaniline-derivatives. Although halogen substitution at the C4-position of an aniline indeed leads to a decreased metabolism at that site, the formation of the reactive 1,4- benzoquinoneimine instead of 4-aminophenol as the primary metabolite, as well as the switch to N-hydroxylation, giving rise to reactive hydroxylamino- and nitroso-derivatives may have considerable toxicological implications.<p>The biological activity of numerous aniline derivatives has been shown to be closely related to the cytochrome P450 mediated oxidative attack at their nitrogen center, yielding products with increasing toxic properties, namely N-hydroxyaniline and nitrosobenzene derivatives. Reactive N-hydroxyanilines cause for example ferrihemoglobin formation (methemoglobinemia) with concomittant co-oxidation to nitrosobenzenes. The resulting nitrosobenzenes in turn are able to interact with cysteine residues of hemoglobin or with the tripeptide glutathione [Eyer, 1988]. On the other hand, the cytochrome P450 mediated aromatic hydroxylation of anilines may represent a detoxification pathway due to the efficient conjugation of the resulting phenolic metabolites and subsequent excretion from the body. However, the cytochrome P450 mediated formation of aminophenol metabolites has been proposed to play a role in the nephrotoxicity of halogenated anilines, occuring predominantly at the proximal tubule and to a lesser extent at the distal tubule [Lo <em>et al.,</em> 1990 & 1991; Rankin <em>et al.,</em> 1986a & b; Valentovic <em>et al.,</em> 1992]. <strong>Chapter 7</strong> presents a clear example of a metabolism-toxicity relationship study. Since the cytochrome P450 mediated regioselective hydroxylation seems to direct the toxicity of aniline compounds, a study was performed on the relationships between the regioselectivity of the hydroxylation of C4-substituted 2-fluoroaniline derivatives and their toxic endpoints nephrotoxicity and/or methemoglobinernia. Depending on the derivative, nephrotoxicity at the tubular site and/or methemoglobinernia was shown to occur. The extent of nephrotoxicity induced by C4-substituted 2-fluoroanilines was shown to be related to the extent of C4-hydroxylation, and the extent of methemoglobinernia was shown to be related to the extent of N-hydroxylation. Consequently, a change in the regioselective hydroxylation from the cytochrome P450-mediated C4-hydroxylation to N-hydroxylation resulted in a change of toxic endpoint from nephrotoxicity to methemoglobinemia.<p>In <strong>chapter 8</strong> a study was directed at understanding the marked differences in biotransformation pathways of anilines and their chemically oxidized analogues nitrobenzenes. <em>In vivo</em> and <em>in vitro,</em> 2,5-difluoroaniline was demonstrated to become predominantly metabolized through a cytochrome P450-mediated pathway, whereas 2,5- dihuoronitrobenzene is predominantly converted through glutathione conjugation, and the a minor extent through nitroreduction and cytochrome P450-mediated aromatic hydroxylation. On the basis of computer calculations a hypothesis was presented that might explain the differences in metabolic pathways on the basis of their molecular orbital substrate characteristics. It was suggested that the HOMO of the thiolate anion of glutathione will interact more efficiently with the LUMO of a nitro compound (E <sub><font size="-1">LUMO</font></sub> = -1.57 eV) than with the LUMO of a less electrophilic amino compound (E <sub><font size="-1">LUMO</font></sub> = -0.08 eV). Concerning the aromatic hydroxylation on the other hand, it was suggested that the SOMO of the cytochrome P450 (FeO) <sup><font size="-2">3+</font></SUP>intermediate can interact more efficiently with the amino compound (E <sub><font size="-1">HOMO</font></sub> = -8.83 eV) than with the nitro compound (E <sub><font size="-1">HOMO </font></sub> = -10.29 eV). These considerations are in accordance with the observation in chapter 4, that the reaction rate of the cytochrome P450-mediated C4-hydroxylation decreases with decreasing electrophilic reactivity of an aniline substrate. Substrates with an E <sub><font size="-1">HOMO </font></sub> value below -9.2 eV were converted at a relatively low rate by the cytochromes P450. The considerations are also in accordance with the recent observation that the rate of both the chemical and glutathione S-transferase catalyzed glutathione conjugation of fluoronitrobenzenes increases with decreasing E <sub><font size="-1">LUMO</font></sub> values [Rietjens <em>et al.,</em> 1995].<p>Halogenated nitrobenzenes are known to become metabolized by nitroreduction, glutathione conjugation and aromatic hydroxylation. For some of the toxic effects of nitrobenzenes, the formation of reactive metabolites is a prerequisite. Reduction of nitrobenzenes to the aminobenzene analogues, via the formation of methemoglobinemia-inducing intermediates (nitrosobenzene, N-hydroxyaniline or radical intermediates), is a clear example of bioactivation of nitrobenzenes. The different susceptibility for nitroreduction, and also the presence of competing biotransformation pathways, like glutathione conjugation and aromatic hydroxylation, have been proposed to set the chances for methemoglobinernia exerted by nitrobenzene-derivatives. Applying QSAR approaches, a study was performed in order to investigate whether a metabolism-toxicity relationship exists for a series of fluoronitrobenzenes providing insight in the relative importance of these two suggestions. <strong>Chapter 9</strong> describes a combined study concerning the influence of the substituent pattern of fluoronitrobenzenes on their biotransformation and their capacity to induce methemoglobinemia. It was demonstrated that increased possibilities for the conjugation of fluoronitrobenzenes to cellular nucleophiles was accompanied by decreased contributions of aromatic hydroxylation and nitroreduction. A QSAR could be described for the rate of conjugation with the tripeptide glutathione or with bovine serum alburnine (a model for cellular nucleophiles) and calculated parameters for electrophilicity of the fluoronitrobenzenes, showing that with increasing number of fluoro-substituents the conjugation pathway will become more important. In addition, the intrinsic reactivity of the fluoronitrobenzenes for nitroreduction by cecal microflora and rat liver preparations was not related to the <em>in vivo</em> methemoglobin-forming capacity. This observation, in combination with the QSAR for conjugation, led to the conclusion that the different methemoglobinemic capacity must rather result from differences in the inherent direct methemoglobinemic reactivity of the various toxic metabolites, and/or from the difference in reactivity of the fluoronitrobenzenes with glutathione or other cellular nucleophiles. As a result of an increased electrophilic reactivity of fluoronitrobenzenes, another toxic endpoint than methemoglobinemia can be expected.<p>In the final study described in <strong>chapter 10</strong> , comparative MO-QSAR studies clearly demonstrated that outcomes on both the regioselectivity and the rate of the cytochrome P450-catalyzed aromatic hydroxylation could be described on the basis of frontier orbital characteristics of the substrates. The MO-QSARs obtained for rats, were also valid for other species, including man. These results strongly support the conclusion that the conversion of the relatively small halogenated benzene derivatives in the relatively large and aspecific active sites of the mammalian cytochromes P450 -even when derived from various species- are mainly dependent on chemical reactivity parameters of the substrates. This importance of the substrate characteristics also dominates over the influence of the specific P450 enzymes in determining the regioselectivity of biotransformation both <em>in vitro</em> and <em>in vivo.</em><p>In conclusion, the studies described in this thesis cleary demonstrate that molecular orbital calculations in combination with the frontier orbital theory are a usefull additional tool to study the mechanism of cytochrome P450 enzyme catalysis. In addition to the theoretical considerations on the molecular reaction mechanisms and enzyme catalysis, empirical data on the metabolism are essential to construct valid rules or even quantitative structure activity relationships for the biotransformation and toxicity of series of related compounds. Insight into the possibilities for biotransformation have been demonstrated to provide insight into the formation of toxicity determining reactive intermediates. Defining QSARs for biotransformation have been shown to offer an approach to explain the type and extent of the toxic effects exerted by the series of halogenated amino- and nitrobenzene derivatives.<p>Refined models, which do not only focus on the chemical reactivity parameters of the substrates, but also take into account possible stereoselective positioning or interactions with specific amino acid residues in the active site, will contribute to a better prediction of metabolic profiles of existing and new drugs, agrochemicals and other industrially relevant compounds.<p>Finally, it is challenging to apply the approaches described in this thesis to other enzymes with more specific active sites than the relatively large aspecific active sites of the biotransformation enzyme cytochrome P450.
The biotransformation of benzene derivatives : the influence of active site and substrate characteristics on the metabolic fate
Koerts, J. - \ 1996
Agricultural University. Promotor(en): C. Veeger; Ivonne Rietjens. - S.l. : Koerts - ISBN 9789054856214 - 197
biotransformatie - cytochroom p-450 - benzeen - derivaten - biotransformation - cytochrome p-450 - benzene - derivatives
<br/>The amount of newly developed chemicals such as agrochemicals, drugs and food additives in our modem society is ever increasing. The industrial production, use and also the release in the environment of these chemicals expose organisms to these xenobiotics. Due to the often hydrophobic character of these xenobiotics they can be easily absorbed and accumulated in the body. However, organisms defend themselves against these agents by converting them enzymatically to more hydrophilic easily excretable products. Sometimes these biotransformation reactions can lead to reactive intermediates which cause cell damage, toxicity, malignancy and/or ultimately carcinogenicity. This indicates why a lot of toxicological screening and safety tests have to be performed during the development of these agents.<p>However, modern society still requires new compounds with better properties. As it is unfeasible to test the biological activity and toxic effects of all newly developed chemicals in experimental animals, many in vitro systems have been developed in which these chemicals can be quickly tested for various biological and/or toxicological effects. To diminish the use of experimental test systems the QSAR concept has been developed, describing so-called Quantitative StructureActivity Relationships. This QSAR approach should make it possible to predict the biological effect, the biotransformation pattern and/or the toxicity of a chemical based on its physical and chemical characteristics. In this respect the application of MO (Molecular Orbital) computer calculations to describe at least some of these chemical characteristics appears to become more and more useful.<p>The present thesis focuses on the use of these computer calculated MO parameters for the prediction of biotransformation characteristics of benzene derivatives. Benzene derivatives play an important role as precursors for the synthesis of many xenobiotics and industrially relevant chemicals. The cytochrome P450-catalysed biotransformation of various substituted benzenes was studied and the results were evaluated to determine whether (MO-based) computer calculated chemical parameters of benzenes could explain the results obtained. Where results could not be described well by the chemical reactivity of the substrate itself, additional experimental work was performed to investigate to which factors and/or interactions the observed deviations could be ascribed. This information can then be used to adopt and improve the predictive model.<p>Biotransformation of xenobiotics by higher organisms is an enzymatic process in which cytochromes P450 play a major role. This system is able to handle an enormous amount of very structurally different chemicals. The cytochrome P450 enzymes differ in their substrate specificity, metabolic activity and their extent of contribution to the production of toxic metabolites. As outlined in the introduction of this thesis <em>(Chapter 1</em> ), there are several factors which control the cytochrome P450-catalysed biotransformation. These factors include for example, electron donation to cytochrome P450, the accessibility of the active (FeO) <sup><font size="-1">3+</font></SUP>species in the active site of the enzyme, the interaction between the substrate and the active site and the chemical reactivity of the substrate itself. In cases where the latter factor controls the outcomes of the cytochrome P450-catalysed biotransformation, one could describe a QSARs for the reaction. Based on these QSARs the cytochrome P450-catalysed biotransformation of other chemicals might be predicted. Furthermore, these QSARs might give more insight in the mechanistic background of the biotransformation reaction for which it has been developed.<p>An example of this approach is described in <em>Chapter 2</em> of this thesis. In this chapter <em>in vivo</em> cytochrome P450-catalysed aromatic ring hydroxylation of various substituted benzene derivatives was determined using <sup><font size="-1">19</font></SUP>F NMR. Previous reported results on the regioselectivity of cytochrome P450-catalysed aromatic ring hydroxylation of a series (poly)fluorobenzenes demonstrated a clear QSAR (correlation 0.96) relating the site of hydroxylation of the site of the reactive HOMO π-electrons in the benzene substrate. Thus, in <em>Chapter 2</em> the same parameter was tested for its ability to predict the regioselectivity of aromatic hydroxylation of a series C4-substituted fluorobenzenes. It turned out that the MO-QSAR approach could well predict the regioselectivity of the series of 4-X-substituted fluorobenzenes when X was a H, F, Cl, and CN group. However, this approach of predicting the regioselectivity of aromatic hydroxylation ran into problems when the <em>para</em> substituent was a bromine or iodine. The extent to which the hydroxylation adjacent to a bromine and iodine substituent was reduced, correlated qualitatively with the size of the substituents as given by their Van der Waals radius, i.e. the larger the substituent the greater the deviation between the predicted and observed hydroxylation at the adjacent aromatic carbon centre. Additional experiments showed that the observed deviations could not be ascribed to a stereoselective orientation of these substrates in the active sites of cytochromes P450. These results led to the hypothesis that bromine and iodine substituents sterically hamper the electrophilic attack of the cytochrome P450 high-valent iron-oxo species on the carbon atoms adjacent to the substituted carbon centres. This hypothesis was supported by calculating the steric effects of bromine and iodine substituents, thus defining a steric correction factor for prediction of the aromatic ring hydroxylation at sites <em>ortho</em> with respect to a bromine or iodine substituent. In a second series of regioselectivity experiments using a series of tri- substituted benzenes containing fluorine, chlorine, bromine, iodine and cyano substituents, these steric correction factors were validated. Without using the steric correction factors for bromine and iodine substituents no correlation between the predicted and observed regioselectivity was obtained. However, upon application of the correction factors a correlation factor of 0.91 was obtained for prediction and actually observed regioselectivity of cytochrome P450-catalysed aromatic hydroxylation of the series tri- substituted benzenes. These results show that the QSAR which was initially developed for a series of (poly)fluorobenzenes was still valid for other substituted benzenes taking into account the steric hindrance by relatively large substituents such as bromine and iodine. Furthermore, from a mechanistic point of view these MO-QSARs suggest that the cytochrome P450-catalysed aromatic ring hydroxylation of at least the benzene derivatives tested proceeds by an initial attack of the iron-oxo species on the ring carbon atom, without the formation of arene oxide intermediates as a major route.<p>The question remains why going from a chlorine to a bromine substituent, steric hindrance is observed rather abrupt. A possible explanation might be found in the observation that when two molecules approach each other the potential energy goes through a minimum and thereafter increases rapidly with decreasing distance.<p>Surprisingly, it was found that steric hindrance by the cyano group was not observed although this group is definitely larger than a bromine. However, with respect to steric hindrance of the electrophilic attack of the (FeO) <sup><font size="-1">3+</font></SUP>species, only the atom closest to the aromatic ring might have to be taken into account. Steric hindrance of this atom, a carbon, is not expected since a carbon, has a size similar to that of a chlorine.<p>More complicated substituents than H, F, Cl, Br, I and CN were investigated in <em>Chapter 3</em> where the regioselectivity of aromatic ring hydroxylation of 3-fluoro(methyl)anilines was investigated. Little is known about the cytochrome P450- catalysed biotransformation of such methylanilines. Results from the <em>in vivo</em> and <em>in vitro</em> cytochrome P450-catalysed regioselectivity of aromatic hydroxylation showed that the frontier orbital density distribution in the aromatic ring could only qualitatively predict the observed regioselectivity. Again, as for the bromine and iodine substituents the observed regioselectivity of aromatic ring hydroxylation for the various amino and methyl substituted substrates showed systematic deviations from the predicted regioselectivity, i.e. hydroxylation at the ring carbon C4 <em>para</em> with respect to the amino moiety was always higher than predicted while hydroxylation at the carbon atoms <em>ortho</em> with respect to the amino group (C2 and C6) was lower than expected. Further research was performed to investigate why for this series of amino and/or methyl substituted benzene derivatives the intrinsic chemical reactivity did not predict the observed regioselectivity of aromatic hydroxylation quantitatively. Three different experimental approaches were used to investigate possible reasons underlying the systematic deviations between predicted and observed regioselectivity for the cytochrome P450-catalysed aromatic hydroxylation of amino-containing benzenes. These results are discussed in some more detail hereafter.<p>1. As a first approach, incubations of 3-fluoro-2-methylaniline with different microsomal preparations containing various cytochrome P450 enzyme patterns as well as with purified cytochrome P4502B1 were performed. The regioselectivity for aromatic hydroxylation of 3- fluoro-2-methylaniline observed in all these incubations were similar. This result indicates that cytochromes P450 with different active sites give similar regioselectivities and, thus, in case of 3-fluoro-2-methylaniline similar deviations for predicted values. This suggests that the observed discrepancy between predicted and observed aromatic hydroxylation patterns can not be ascribed to a stereoselective orientation of the 3-fluoro(methyl)anilines imposed by the active sites of the cytochromes P450 catalysing its conversion.<p>2. Further support for this conclusion was obtained from <sup><font size="-1">1</font></SUP>H NMR T <sub><font size="-1">1</font></sub> relaxation studies on fluoromethylanilines. These studies are described in <em>Chapter 4.</em> It appears that the orientation of the tested fluoromethylanilines in the active sites of cytochromes P4501AI and 2B1 is similar. Based on the observation that all aromatic protons are at about the same average distance from the catalytic Fe <sup><font size="-1">3+</font></SUP>centre, these results are most compatible with a time-averaged orientation of the substrates with the Fe <sup><font size="-1">3+</font></SUP>above the aromatic ring and the π-orbitals of the aromatic ring and those of the porphyrin rings in a parallel position. This latter aspect would provide possibilities for energetically favourable π-πinteractions. Possibilities for a flip-flop rotation around an axis in the plane of the aromatic ring of the substrate can be included in this picture, as such rotations would still result in a similar average distance of all aromatic protons to the Fe <sup><font size="-1">3+</font></SUP>centre. Altogether, the data strongly suggest that, independent of the cytochrome P450 enzyme these substrates can rotate freely in the active site and are not hindered and/or specifically orientated by interactions with specific amino acid residues of the active site.<p>3. Finally, the possible influence of the active site on the cytochrome P450-catalysed aromatic hydroxylation was tested by using microperoxidase-8 as a model system for cytochrome P450-catalysed aromatic ring hydroxylation. Microperoxidase-8 is a so-called mini-enzyme prepared by proteolytic digestion of horse-heart cytochrome c. MP-8 contains a heme covalently attached to a peptide chain of only eight amino acids and thus it lacks an active site. MP-8 is well known to perform peroxidase-like reactions. However, experimental evidence has been obtained described in <em>Chapter 5</em> of this thesis that the MP-8-catalysed aromatic ring hydroxylation of aniline and phenol derivatives in a H <sub><font size="-1">2</font></sub> O <sub><font size="-1">2</font></sub> -driven system proceeds by a cytochrome P450-like oxygen-transfer mechanism. This could be concluded from the fact that <sup><font size="-1">18</font></SUP>O incorporation from H<font size="-1"><sub>2</sub><sup>18</SUP></font>O <sub><font size="-1">2</font></sub> into the 4-aminophenol formed from aniline was 100%. Thus, the H <sub><font size="-1">2</font></sub> O <sub><font size="-1">2</font></sub> -driven MP-8 system was used as a model to study regioselectivity of oxygen-transfer in a heme-based catalyst without an active site.<p>The results of such experiments demonstrated that the MP-8-catalysed aromatic ring hydroxylation of 3-fluoro(methyl)anilines <em>(Chapter 3)</em> results in similar regioselectivities of aromatic ring hydroxylation as those observed for cytochrome P450- catalysis. As a specific substrate binding site in MP-8 can be excluded, these results, together with those obtained from different microsomal preparations <em>(Chapter 3)</em> and the <sup><font size="-1">1</font></SUP>H NMR T <sub><font size="-1">1</font></sub> relaxation measurements <em>(Chapter 4),</em> clearly eliminates the possibility that the regioselectivity of aromatic ring hydroxylation is predominantly dictated by a stereoselective orientation of the substrate in the active site of cytochromes P450. Thus for these relatively small and non-polar substrates specific interactions between molecular sides in the substrate and amino acid side chains present in the active site are not dictating a stereoselective orientation of these substrates.<p>Since the MP-8-catalysed reactions showed similar deviations between predicted and observed regioselectivity of aromatic hydroxylation of 3-fluoro(methyl)anilines, it was concluded that the deviations must result from an orientating interaction between the substrate and the high-valent iron-oxo species (FeO) <sup><font size="-1">3+</font></SUP>itself, resulting in a stereoselective orientation of the substrate towards the catalytically active species. This interaction can be expected to be quite similar for different cytochromes P450 and might also be comparable in the active MP-8 system. This implies that, in contrast to what was observed by <sup><font size="-1">1</font></SUP>H NMR for substrates bound to the Fe <sup><font size="-1">3+</font></SUP>resting state of the enzyme, the (FeO) <sup><font size="-1">3+</font></SUP>form of the protein would impose a preferential orientation of the amino-containing substrate in such a way that on the average C4 ( <em>para</em> to the amino) is closer to whereas <em>C2/6 (ortho</em> to the amino) are further away from the (FeO) <sup><font size="-1">3+</font></SUP>species. Support for a different substrate orientation in the Fe <sup><font size="-1">3+</font></SUP>resting state and the (FeO) <sup><font size="-1">3+</font></SUP>activated form respectively can be found in a study of Paulsen and Ornstein. They showed that the orientation of camphor in the presence and absence of the high-valent iron-oxo species (FeO) <sup><font size="-1">3+</font></SUP>was different and that orientation in the presence of the catalytically active form of the enzyme (FeO) <sup><font size="-1">3+</font></SUP>was in accordance with the observed regioselectivity of oxidation.<p>Altogether, results from all studies on the regioselectivity of aromatic hydroxylation of the various benzene derivatives show that the regioselectivity of the H, F, Cl, Br, I, CN, CH <sub><font size="-1">3</font></sub> and NH <sub><font size="-1">2</font></sub> substituted benzene derivatives investigated is predominantly determined by their chemical reactivity. However, especially for amino-containing benzene derivatives interactions between the substrate and the high-valent iron-oxo species, resulting in a stereoselective orientation of the substrate, might also play a role. This means that the QSAR for aromatic ring hydroxylation in the case of amino-containing benzenes predicts only qualitatively the regioselectivity of aromatic ring hydroxylation for these series of substrates.<p>An additional result obtained in all regioselectivity studies was the absence of formation of NIH shifted phenolic metabolites. The formation of such NIH shifted phenolic metabolites has been well documented especially for the biotransformation of chlorinated and brominated benzene derivatives. As the benzenes in our studies were mainly fluorinated, we decided to investigate whether there was a (chemical) reason for this discrepancy.<p><em>In Chapter 6</em> the possibility of a cytochrome P450-catalysed fluorine NIH shift in a series of polyfluorobenzenes was investigated and compared with the literature data of the chlorinated analogues. The <em>in vivo</em> biotransformation of a series polyfluorobenzenes showed that formation of NIH shifted metabolites was not a significant biotransformation route. As outlined above, this is in contrast to the results reported in the literature for the chlorinated analogues. However, in contrast to the <em>in vivo</em> data, in <em>in vitro</em> microsomal studies with 1,4-difluorobenzene formation of a significant amount of fluorine NIH shifted phenolic metabolite was observed. Unfortunately, the <em>in vitro</em> microsomal conversion of the other used polyfluorobenzenes could not be detected, possibly due to the fact that the HOMO energy of these substrates was too low for an efficient conversion by cytochromes P450 . It is generally accepted that formation of NIH shifted metabolites proceeds via arene oxide intermediates and that these arene oxides, especially <em>in vivo,</em> but not <em>in vitro</em> in a microsomal system, might react in competing conjugation pathways such as GSH conjugation. Additional <em>in vivo</em> and <em>in vitro</em> experiments showed that indeed GSH conjugation was a competing pathway for the arene oxide intermediates, its presence or absence resulting in an influence on the amount of NIH shifted phenolic metabolites observed.<p>Nevertheless, the results thus obtained also clearly illustrated and confirmed the reduced capacity for formation of NIH shifted phenolic metabolites for fluoro- as compared to chlorobenzenes. This differences between the NIH shift for fluorine and chlorine containing benzenes was further investigated and could be explained by MO computer calculations on the proposed reaction intermediates in the two biotransformation pathways. Epoxide ring opening, supposed to be the rate-limiting step in the formation of NIH shifted metabolites, appeared easier for chlorinated benzenes compared to their fluorinated analogues. Furthermore, these MO calculations in combination with a MO-QSAR for the rate of GS-conjugation of electrophilic model compounds were indicative for higher rates of GSH conjugation for the fluorinated benzene arene oxides than the chlorinated analogues. The detoxifying pathway of GSH conjugation is very important for assessing the risks of these kind of substrates as arene oxides are known to react with protein and DNA provoking toxic and/or mutagenic /carcinogenic responses.<p>Altogether, the results from this study show that MO calculations can not only explain and thus predict the regiospecificity of biotransformation in a molecule but also explain the relative rates, i.e. chances, on different biotransformation routes.<p>Finally, in addition to all studies on model benzene derivatives, the last chapter of this thesis <em>(Chapter 7)</em> describes studies on the biotransformation of a halogenated benzene derived compound that is used as an insecticide, i.e. teflubenzuron. This may provide some insight in the possibilities to use outcomes from studies on model compounds to obtain insights also in the biotransformation of other compounds. Teflubenzuron, which is used for the protection of fruit and vegetables against larves, contains two aromatic benzene-derived rings connected via an urea bridge. These aromatic rings are differently substituted and are a suitable starting point to evaluate the influence of various substituents on the metabolic fate of the chemical. Basically, they are an aniline and a benzoate derived moiety. Results of this study on the biotransformation of teflubenzuron demonstrate that upon an oral dose of the insecticide to male Wistar rats the metabolic fate of the two aromatic rings is significantly different. Though about 90% of the dose was excreted unchanged in the faeces, the remainder of the dose was absorbed from the gastrointestinal tract and -in part- excreted in the urine mainly after hydrolysis of the urea bridge. Interestingly, the urinary recovery of the benzoate moiety was about 8 times higher than that of the aniline moiety of teflubenzuron. Dose-recovery studies on the scission products of teflubenzuron confirmed these results as the urinary recovery of the metabolites from the benzoate moiety were nearly 100% while the recovery of the aniline derivatives was only about 50%. The benzoate moiety is excreted unchanged or after a Phase IIreaction, while the aniline moiety has to undergo both a Phase I and II reaction before it can be excreted.The significant difference between the recovery of the benzoate and aniline moiety can be best explained by the formation of a cytochrome P450-catalysed reactive benzoquinoneimine from the C4 halogenated aniline derivatives. Swift binding of these reactive benzoquinoneimines to tissue macromolecules might cause the compound to be withheld in the body. This is also important from a toxicological point of view as binding of these reactive benzoquinoneimines might provoke toxic and/or mutagenic /carcinogenic effects in the body.<p>Altogether, the results described in this thesis show that QSARs are very useful in explaining the biotransformation pattern of relatively small and non-polar benzene derivatives. Their biotransformation pattern was demonstrated to be predominantly determined by the chemical reactivity of the benzenes and - although to a much lesser extent for bromine, iodine and amino containing derivatives- more than by steric: factors (Br,I) and/or electronic dipole-dipole interactions with the activated (FeO) <sup><font size="-1">3+</font></SUP>cofactor (NH <sub><font size="-1">2</font></sub> ). However, the further extension of the presently developed QSARs to more complicated and especially to larger molecules might require the incorporation of computer techniques that take into account preferential stereoselective substrate orientations imposed by the active sites. Although the molecular modelling computer techniques to do these type of additional orientation analyses are available, such studies also require the availability of the enzyme crystal structures. Since such 3-D structures for cytochromes P450 are at present restricted to some bacterial cytochromes P450 and not available for mammalian cytochromes P450 the studies may have to await the elucidation of the mammalian cytochrome P450 structures. Nevertheless, the concept as such might be developed using either molecular modelling combined with quantum chemical calculations for the bacterial cytochrome P450 system as done recently by Zakharieva et al. and Freutel et al., or by performing similar studies for other biotransformation enzymes, such as for example the glutathione S-transferases for which several 3-D structures have been described.<p>In general, QSARs can be used to predict the biological activity of non-tested chemicals but they can also give more insight in the mechanistic rules of biotransformation of molecules. This knowledge can be very helpful for example when the technical ability of cloning cytochromes P450, and thus producing these enzymes in large quantities, is increasing. Cytochromes P450 could then be used for the synthesis of chemical products (P450 biotechnology). Especially cytochromes P450 are suitable as they are able to convert many structural diverse substrates. The advantage of enzymatic synthesis compared to chemical synthesis is that it causes among others less waste problems.
|Biosorptie van chloorbenzenen aan mineraliserende algen.
Koelmans, A.A. - \ 1994
In: Kontaminanten in bodems en sediment : sorptie en biologische beschikbaarheid : een symposium, 29 april 1993, De Reehorst, Ede / Evers, E., Opperhuizen, A., Voorend, L., - p. 65 - 72.
aquatische gemeenschappen - plantengemeenschappen - derivaten - benzeen - chloride - biologische beschikbaarheid - adsorptie - sorptie - toxicologie - algen - ecotoxicologie - bioaccumulatie - aquatic communities - plant communities - derivatives - benzene - bioavailability - adsorption - sorption - toxicology - algae - ecotoxicology - bioaccumulation
Sorption of chlorobenzenes to mineralizing phytoplankton.
Koelmans, A.A. ; Sanchez-Jimenez, C. ; Lijklema, L. - \ 1993
Environmental Toxicology and Chemistry 12 (1993). - ISSN 0730-7268 - p. 1425 - 1439.
plankton - adsorptie - sorptie - benzeen - derivaten - adsorption - sorption - benzene - derivatives
Versatility of soil column experiments to study biodegradation of halogenated compounds under environmental conditions.
Meer, J.R. van der; Bosma, T.N.P. ; Bruin, W.P. de; Harms, H. ; Holliger, C. ; Rijnaarts, H.H.M. ; Tros, M.E. ; Schraa, G. ; Zehnder, A.J.B. - \ 1992
Biodegradation 3 (1992). - ISSN 0923-9820 - p. 265 - 284.
benzeen - chloride - derivaten - hexachloorbenzeen - gehalogeneerde koolwaterstoffen - microbiële afbraak - pseudomonas - bodemschimmels - biodegradatie - benzene - derivatives - hexachlorobenzene - halogenated hydrocarbons - microbial degradation - soil fungi - biodegradation
The relationship between biotransformation and toxicity of halogenated benzenes : nature of the reactive metabolites and implications for toxicity
Besten, C. den - \ 1992
Agricultural University. Promotor(en): J.H. Koeman, co-promotor(en): Peter van Bladeren. - S.l. : Den Besten - ISBN 9789054850465 - 215
benzeen - derivaten - gehalogeneerde koolwaterstoffen - biotransformatie - cytochroom p-450 - toxiciteit - benzene - derivatives - halogenated hydrocarbons - biotransformation - cytochrome p-450 - toxicity
Lipofiele xenobiotica worden in het lichaam door middel van biotransformatie omgezet in polaire metabolieten, zodat excretie via urine enlof faeces mogelijk wordt. In de meeste gevallen resulteert biotransformatie in de ontgifting van toxische stoffen. Er zijn echter vele stoffen bekend die op zichzelf weinig schadelijk zijn, maar juist als gevolg van biotransformatie worden omgezet in reactieve produkten (zgn. 'metabole activatie'), die vervolgens de normale fysiologie van een cel, weefsel of organisme kunnen verstoren. Voor een beter inzicht in de relatie tussen chemische structuur van een stof en zijn effect(en) op levende organismen is kennis van de verschillende routes, waarlangs een stof wordt omgezet, onontbeerlijk.
In hoofstuk 1 wordt een beknopte beschrijving gegeven van de belangrijkste biotransformatie systemen, die betrokken zijn bij de omzetting van xenobiotica, en van de bijdrage die deze systemen kunnen leveren aan de metabole activering enlof toxiciteit van xenobiotica. Gehalogeneerde benzenen vormen een goed voorbeeld van een groep van stoffen, waarbij metabole activatie een rol speelt in de toxische effecten op de verschillende doelorganen (met name lever en nier). Figuur 1.2 (zie pagina 33) maakt duidelijk dat uit een betrekkelijk eenvoudige stof, zoals een halogeenbenzeen, verschillende reactieve intermediairen en metabolieten gevormd kunnen worden. Het vele onderzoek naar het mechanisme van levertoxiciteit door broombenzeen wijst op een belangrijke rol voor het intermediair 3,4-epoxide. Echter, tijdens de oxidatie van het volledig gesubstitueerde hexachloorbenzeen zijn het niet de epoxides, maar juist reactieve chinon metabolieten (met name tetrachloorbenzochinon), die covalent aan eiwit binden. Daarnaast zijn er sterke aanwijzigingen dat de niertoxiciteit door broombenzeen is toe te schrijven aan de vorming van chinon metabolieten, die na conjugatie met glutathion naar de nier getransporteerd worden en daar proximale tubulaire schade veroorzaken.
Het onderzoek dat staat beschreven in dit proefschrift is uitgevoerd om meer inzicht te verkrijgen in het belang van de verschillende intermediairen en metabolieten in de toxiciteit van een hele serie gehalogeneerde benzenen. Experimenten zijn met name uitgevoerd met (poly-)chloorbenzenen. Enerzijds zijn er in vitro studies gedaan met [ 14C]-gelabelde substraten en microsomale suspensies om de verschillende biotransformatie routes te karakterizeren en het belang van deze routes in de metabole activatie te evalueren (Deel I, hoofdstuk 2, 3, 4, en 5; samengevat in hoofdstuk 6). Anderzijds zijn in vivo studies uitgevoerd om (i) een inschatting te krijgen van de toxiciteit van een reeks chloorbenzenen op de verschillende doel organen, en (ii) meer inzicht te krijgen in de rol van de verschillende metabolieten in de toxische effecten (Deel II, hoofdstuk 7, 8, en 9; samengevat in hoofdstuk 10).
Deel I In vitro studies
In het eerste deel van dit proefschrift komt de vraag aan de orde óf, en zo ja in welke mate, een serie polychloorbenzenen geoxideerd kan worden tot reactieve produkten die covalent aan eiwit kunnen binden. Hiertoe werden microsomale incubaties met [ 14C]-gelabelde substraten uitgevoerd.
In hoofdstuk 2 wordt de microsomale oxidatie van pentachloorbenzeen (PCB) naar pentachloorfenol beschreven. Daarnaast worden de verschillende isomeren van tetrachloorfenol gevormd alsmede tetrachloorhydrochinon. Tijdens de oxidatie van PCB worden metabolieten gevormd, die covalent binden aan eiwit. Incubaties uitgevoerd in de aanwezigheid van ascorbine zuur, een reductor, tonen het belang van chinon metabolieten in de eiwit binding aan. Vanwege het feit dat de eiwitbinding ondanks een grote overmaat ascorbine zuur nooit geheel voorkomen kan worden, wordt de suggestie gedaan dat andere metabolieten, waarschijnlijk de epoxides in de primaire oxidatie van PCB naar pentachloorfenol, ook een bijdrage leveren aan de eiwitbinding.
Hoofdstuk 3 beschrijft de microsomale oxidatie van 1,2,4-trichloorbenzeen (1,2,4-TRICB). 1,2,4-TRICB wordt geoxideerd tot de verschillende isomeren van trichloorfenol en, in mindere mate, tot trichloorhydrochinon. De vorming van de 2,3,4- en 2,4,6-trichloorfenol kan verlopen via een zgn. 'NIH-shift' van de overeenkomstige epoxide intermediairen in de primaire oxidatie stap. In overeenkomst met het microsomaal metabolisme van PCB blijkt een belangrijk deel van de metabolieten covalent aan eiwit te binden. Opvallend is echter dat de eiwitbinding van 1,2,4-TRICB metabolieten volledig voorkomen wordt door de aanwezigheid van ascorbine zuur in de microsomale incubatie. Daarnaast blijkt dat zowel voor 1,2,4-TRICB als voor PCB de mate van eiwit binding sterk gecorreleerd is met de mate van secundaire oxidatie van de fenol metabolieten, en niet met de mate van primaire oxidatie van de benzenen. Op grond van deze waarnemingen wordt geconcludeerd dat chinon metabolieten de enige reactive species zijn in de oxidatie van zowel 1,2,4-TRICB als PCB. In plaats van de aanname dat de 'rest' binding van PCB metabolieten aan eiwit in de aanwezigheid van ascorbine zuur (zie hoofdstuk 2) veroorzaakt wordt door een ander type reactieve metaboliet, kan als verklaring dienen een onvolledige reductie van het reactieve tetrachloorbenzochinon tot tetrachloorhydrochinon.
Hoofdstuk 3 beschrijft ook de invloed van de cytochroom P450 samenstelling van de microsomale suspensies op de oxidatie van PCB en 1,2,4-TRICB. Microsomen van ratten voorbehandeld met dexamethason (DEX), een stof die de hoeveelheid P450IIIA1 sterk induceert, zetten de beide substraten het beste om, in analogie met wat gevonden was voor hexachloorbenzeen (HCB) (Van Ommen et al. , 1989). Het blijkt echter dat met afnemende substitutie graad de bijdrage van P450IIIA1 aan de oxidatie minder prominent wordt, en de relatieve bijdrage van andere isoenzymen belangrijker. De microsomale studies met twee isomeren van dichloorbenzeen bevestigen deze tendens (zie hoofdstuk 4).
Van twee dichloorbenzeen isomeren (DCB; i.e. 1,2- en 1,4-DCB) is een groot verschil in hepatotoxiciteit beschreven (zie ook hoofdstuk 7). In hoofdstuk 4 wordt de microsomale oxidatie van de toxische 1,2-isomeer en de niet-toxische 1,4-isomeer beschreven, met speciale aandacht voor mogelijke verschillen in metabolieten profiel die een rol kunnen spelen in de isomeer-selectieve hepatotoxiciteit. Zowel 1,2- als 1,4-DCB worden geoxideerd tot metabolieten die covalent aan eiwit binden. Een interessante waarneming is echter dat de eiwitbinding van 1,4-DCB metabolieten door de reductor ascorbine zuur volledig geremd kan worden, terwijl dit voor 1,2-DCB slechts gedeeltelijk mogelijk is. 'Molecular Orbital' computer berekeningen aan de arene oxide/ oxepin intermediairen van beide substraten tonen echter geen verschillen aan in chemische reactiviteit enlof stabiliteit van de verschillende arene oxide intermediairen van 1,2-DCB versus 1,4-DCB. Dit pleit tegen een rol van arene oxide intermediairen in de ('rest') eiwit binding van 1,2-DCB.
Vooruitlopend op hoofdstuk 5, waarin het mechanisme van de vorming van reactieve chinon metabolieten wordt bestudeerd, wordt het optreden van restbinding van 1,2-DCB metabolieten aan eiwit in aanwezigheid van een reductor verklaard uit een directe oxidatie van de para -gesubstitueerde fenol metaboliet (i.e., 3,4,-dichloorfenol) naar het reactieve benzochinon. Een dergelijke directe oxidatie van de primaire fenol metaboliet van 1,4-DCB (i.e. 2,5-dichloorfenol; bezit geen halogeen substituent para ten opzichte van de hydroxyl groep) is niet mogelijk.
Bovenstaande studies benadrukken het belang van de vorming van secundaire chinon metabolieten in de metabole activatie van gechloreerde benzenen. Het is daarom van belang om het mechanisme van vorming van deze reactieve produkten te bestuderen. Hiertoe worden pentafluor- en pentachloorfenol, en hun niet para gesubstitueerde analogen gebruikt als model stoffen. De oxidatie produkten in een microsomale incubatie worden geïdentificeerd met 19F-NMR en HPLC. Het karakter van de oxidatie produkten van de fenol substraten kan echter niet bestudeerd worden onder standaard condities met moleculaire zuurstof en NADPH als electronen donor voor cytochroom P450, vanwege het feit dat het aanwezige NADPH eventueel gevormd benzochinon (chemisch) kan reduceren tot hydrochinon. Daarom zijn anaerobe incubaties uitgevoerd, waarbij cumeen hydroperoxide werd gebruikt als zuurstof donor. Gebaseerd op de resultaten die beschreven staan in hoofdstuk 5, wordt een mechanisme voorgesteld voor de P450 afhankelijke oxidatie van halogeenfenolen, waarbij het al dan niet aanwezig zijn van een halogeensubstituent op de positie para ten opzichte van de hydroxyl groep een bepalende factor is voor het karakter van het produkt (i.e. hydrochinon vs benzochinon). Cytochroom P450 afhankelijke oxidatie op een niet-gesubstitueerde para positie resulteert in de vorming van het para -hydroxyl derivaat (i.e. hydrochinon) als primair produkt. Daarentegen resulteert cytochroom P450 afhankelijke oxidatie op een para positie waar een halogeen substituent zit in de directe vorming van een reactief benzochinon onder verlies van het halogeen als een halogeen anion. In Hoofdstuk 11 wordt ingegaan op mogelijke toxicologische implicaties van directe vorming van reactieve benzochinon metabolieten.
Deel II In vivo studies
In deel II van dit proefschrift staan de resultaten van twee in vivo studies beschreven, waarbij de toxiciteit van een hele serie chloorbenzenen op de verschillende doel organen is vastgesteld. Tevens wordt getracht de diverse effecten te correleren met de vorming van verschillende metabolieten.
In Hoofdstuk 7 wordt een hele serie chloorbenzenen vergeleken met betrekking tot hun potentie om toxiciteit te veroorzaken in verschillende doel organen na een éénmalige i.p. toediening in de rat. Effecten op de lever bestaan uit een toegenomen relatief levergewicht op 72 uur na toediening, hetgeen voor de hoger gechloreerde benzenen ook nog waarneembaar is na 216 uur. In de meeste behandelingsgroepen wordt een variabele mate van centrilobulaire hypertrofie en hepatocellulaire degeneratie waargenomen. Van alle congeneren die getest zijn, geven 1,2-dichloorbenzeen en 1,2,4- trichloorbenzeen de meest ernstige effecten, hetgeen blijkt uit een sterke stijging van de plasma ALT spiegels, en duidelijk waarneembare histopathologische degeneratieve veranderingen. Monochloorbenzeen was minder toxisch. In deze studie worden geen schadelijke effecten op de lever waargenomen na blootstelling aan 1,4-dichloorbenzeen en 1,2,4,5-tetrachloorbenzeen, terwijl blootstelling aan pentachloorbenzeen resulteert in slechts geringe histopathologische veranderingen in de lever. Hieruit kan dus geconcludeerd worden dat de mate van metabole activatie zoals waargenomen in vitro (deel I) geen goede weergave biedt van de potentie om leverschade te veroorzaken.
Er worden in de gebruikte dosis range geen degeneratieve effecten waargenomen op de nieren. Wel blijkt de vorming van 'Protein Droplets' in de tubulaire epitheel cellen een algemeen verschijnsel te zijn na (éénmalige) blootstelling aan gechloreerde benzenen. Dit effect was sterker en gedurende langere tijd waarneembaar na blootstelling aan de hoger gechloreerde congeneren.
Een interessante waarneming betreft het feit dat chloorbenzenen de schildklierhormoon huishouding verstoren, hetgeen tot uiting komt in een sterke daling van de plasma thyroxine spiegels. Hoger gechloreerde benzenen veroorzaken een sterker daling, en deze daling blijkt tevens op te treden na subchronische toediening van een lage dosis (hoofdstuk 8). Verstoring van de schildklierhormoon huishouding is ook beschreven voor de polychloorbiphenylen (Brouwer, 1989), verbindingen die wat betreft chemische structuur grote overeenkomsten vertonen met chloorbenzenen. Er worden in hoofdstuk 7 aanwijzingen geleverd voor een gemeenschappelijk werkingsmechanisme, waarbij de vorming van fenol metabolieten een cruciale factor is: door een selectieve interactie van fenol metabolieten met transthyretiene, een belangrijk plasma transport eiwit voor schildklierhormoon in de rat, wordt het plasma transport van thyroxine ernstig verstoord, hetgeen resulteert in verlaging van de plasma spiegels.
In hoofdstuk 8 en 9 worden de resultaten beschreven van een 13-weken dieet studie in de rat (♀) met hexachloorbenzeen (HCB) en pentachloorbenzeen (PCB). Vergelijking van de toxiciteit en van de biotransformatie van deze twee congeneren is interessant, aangezien in vitro studies hadden aangetoond dat zowel HCB als PCB geoxideerd worden tot pentachloorfenol (PCP) en het reactieve tetrachloorbenzochinon (TCBQ) (zie Van Ommen et al., 1986; hoofdstuk 2). Daarnaast zijn er sterke aanwijzing dat hetzelfde cytochroom P450 isoenzym (P450IIIA) betrokken is bij de omzetting van deze stoffen (Van Ommen et al., 1989; hoofdstuk 3).
De subchronische studie beschreven in hoofdstuk 8 was met name gericht om meer inzicht te krijgen in de rol van de oxidatieve metabolieten in de diverse toxische effecten van HCB en PCB. Bijzondere aandacht was er voor de mogelijke rol van het reactieve TCBQ in het ontstaan van porfyrie. Hiertoe werd de excretie van PCP en tetrachloorhydrochinon, de gereduceerde vorm van TCBQ, in de urine gevolgd in de tijd. Daarnaast werd het effect bestudeerd van selectieve beinvloeding van de cytochroom P450IIIA activiteit op de toxiciteit (met name porfyrie) en de biotransformatie van HCB en PCB, door gelijktijdige behandeling met het macrolide triacetyloleandomycine (TAO).
Blootstelling van ratten (♀) aan HCB (300 ppm) via het voer resulteert in een sterke toename van de porfyrine excretie via de urine en in een sterke accumulatie van porfyrines in de lever. Deze effecten van HCB worden sterk geremd door gelijktijdige blootstelling aan TAO. Gecombineerde blootstelling van ratten aan HCB of PCB met TAO resulteert tevens in een duidelijk verlaagde uitscheiding van de oxidatieve metabolieten, PCP en TCHQ. De vermindering van de porfyrinogene effecten van HCB kunnen echter niet verklaard worden uit een verminderde vorming van het reactieve TCBQ, aangezien ratten die worden behandeld met een hoge dosis PCB (1300 ppm) een urinaire excretie van TCHQ hebben die enkele malen hoger is dan in ratten die het HCB dieet krijgen, terwijl deze ratten géén porfyrie ontwikkelen. De goede correlatie die werd waargenomen tussen porfyrie en PCP excretie kunnen echter wel de hypothese ondersteunen, waarin een reactief intermediair in de primaire oxidatie stap van HCB naar PCP wordt voorgesteld als uiteindelijke porfyrinogene species. In dit opzicht is met name een nieuw gepostuleerd type (reactief) intermediar met een chinon-structuur (Rietjens en Vervoort, 1992) interessant voor verder onderzoek.
In hoofdstuk 9 wordt de biotransformatie van HCB en PCB beschreven, waarin de metabolieten profielen in de urine worden vergeleken. Zoals reeds besproken in hoofdstuk 8, worden HCB en PCB in vivo geoxideerd tot PCP en TCHQ. Dit blijken echter de enige twee gemeenschappelijke metabolieten van HCB en PCB, die worden uitgescheiden via de urine. Overige metabolieten van HCB zijn het pentachloorfenylmercaptuurzuur, kwantitatief de belangrijkste metaboliet, en mercaptotetrachloorthioanisool. PCB wordt omgezet in een groter aantal zwavelbevattende verbindingen, waarbij mercaptotetrachloorfenol en pentachloorthiofenol de belangrijkste produkten zijn.
PCB wordt, behalve tot PCP, in belangrijke mate ook geoxideerd tot 2,3,4,5-tetrachloorfenol (TCP). Opvallend is dat de excretie van 2,3,4,5-TCP niet geremd wordt door gecombineerde blootstelling van ratten aan PCB en TAO, de remmer van P450IIIA. Dit wijst erop dat (i) de oxidatie van PCB tot PCP en tot 2,3,4,5-TCP verloopt via verschillende routes, en dat (ii) cytochroom P450IIIA niet betrokken is bij de oxidatie van PCB tot 2,3,4,5-TCP. Gecombineerde blootstelling van PCB en TAO had geen éénduidig effect op de excretie van zwavelbevattende metabolieten. Zo blijkt bijvoorbeeld dat de excretie van mercaptotetrachloorfenol niet geremd wordt door TAO, terwijl die van (het glucuronide van) pentachloorthiofenol sterk geremd wordt. De resultaten van deze studie onderstrepen het belang van meer gedetailleerde studies naar de interactie tussen fase I metabolisme (oxidatieve systemen, met name cytochroom P450) en fase II metabolisme (conjugatieve systemen, met name glutation conjugatie) om uiteindelijk een beter begrip te krijgen van de routes waarlangs de verschillende metabolieten gevormd worden.
Degradation of benzene compounds by yeasts in acidic soils.
Middelhoven, W.J. ; Koorevaar, M. ; Schuur, G.W. - \ 1992
Plant and Soil 145 (1992). - ISSN 0032-079X - p. 37 - 43.
benzeen - derivaten - microbiële afbraak - microbiologie - organische verbindingen - bodem - bodemchemie - bodemschimmels - benzene - derivatives - microbial degradation - microbiology - organic compounds - soil - soil chemistry - soil fungi
|Enrichment and properties of an anaerobic mixed culture reductively dechlorinating 1,2,3-trichlorobenzene to 1,3-dichlorobenzene.
Holliger, C. ; Schraa, G. ; Stams, A.J.M. ; Zehnder, A.J.B. - \ 1992
Applied and Environmental Microbiology 58 (1992). - ISSN 0099-2240 - p. 1636 - 1644.
anaërobe micro-organismen - bacteriën - benzeen - derivaten - degradatie - metabolisme - micro-organismen - organische chloorverbindingen - anaerobes - bacteria - benzene - derivatives - degradation - metabolism - microorganisms - organochlorine compounds
Molecular mechanisms of adaptation of soil bacteria to chlorinated benzenes
Meer, J.R. van der - \ 1992
Agricultural University. Promotor(en): A.J.B. Zehnder; W.M. de Vos. - S.l. : Van der Meer - 100
bodembacteriën - pseudomonas - derivaten - benzeen - chloride - hexachloorbenzeen - micro-organismen - genetica - heritability - adaptatie - soil bacteria - derivatives - benzene - hexachlorobenzene - microorganisms - genetics - adaptation - cum laude
<p>The pollution of our environment with a large number of different organic compounds poses a serious threat to existing life, since many of these chemicals are toxic or are released in such quantities that exceed the potential of biological detoxification and degradation systems. Bacteria and other microorganisms play an essential role in the breakdown of xenobiotic compounds. Microbes use these compounds as carbon and energy source and metabolize them to harmless endproducts. However, not all compounds are easily metabolized and some structures resist the action of existing enzyme systems in bacteria. Nevertheless, bacterial species have been isolated which have overcome these metabolic barriers and completely metabolize chemicals that were previously considered to be persistent.<p>The project of this thesis was initiated to study the genetic mechanisms in bacteria that cause adaptation to use xenobiotic compounds as novel growth substrates (see Chapter I for a review). The work presented here mainly focused on one class of compounds, i. e. lower chlorinated benzenes such as dichlorobenzenes (DCB) and 1,2,4- trichlorobenzene (1,2,4-TCB). These compounds were known to be very resistant to biodegradation by bacteria. A number of bacterial species was isolated by enrichment techniques which were able to use DCB's and/or 1,2,4-TCB as sole carbon and energy source for growth. One of these bacteria, <em>Pseudomonas</em> sp. strain P51, was investigated further in this study. We have obtained strong evidence that the pathway for chlorobenzene metabolism arose as a consequence of the unique combination of two gene clusters, each specifying part of the complete pathway. These individual gene clusters are not uncommon and probably exist separately in different bacteria. Our results suggest that one of the gene clusters is contained in a novel transposable element that may have been acquired by strain P51 and integrated into a catabolic plasmid that already contained the other gene cluster. A further fine-tuning of the new pathway may have occurred through specialization of individual enzymes towards novel metabolic intermediates and by changes in the regulatory system in response to novel inducer molecules.<p>The degradation of DCB's and 1,2,4-TCB was studied at concentrations between 10 μg/l and 1 mg/l in soil percolation columns filled with sediment of the Rhine river, which in some cases were inoculated with <em>Pseudomonas</em> sp. <em></em> strain P51 (Chapter 2). In the inoculated columns, DCB's and 1,2,4-TCB were instantly degraded. Strain P51 remained viable in the column as long as the chlorinated benzenes were fed in the influent. Interestingly, minimal concentrations of the chlorinated benzenes were measured in the effluent of the columns, independently of the influent concentrations used (6 ± 4 μg/l for 1,2-DCB; 20 ± 5 μg/l for 1,2,4-TCB; more than 20 μg/l for 1,3-DCB and 1,4-DCB), which could not be lowered by additional inoculations with strain P51. The native microbial population in the noninoculated columns adapted to degrade 1,2-DCB after a lag phase of about 60 days, and was then able to remove a concentration of 25 μg/l of 1,2-DCB in the influent to less than 0.1 μg/l.<p>Detailed genetic studies were carried out with <em>Pseudomonas</em> sp. <em></em> strain P51 to characterize the genetic determinants for chlorobenzene metabolism. A large plasmid of 110 kilobase-pairs (kb) (pP51) could be isolated from cells that were cultivated on 1,2,4- TCB (Chapter 3). This plasmid could be cured from the strain by successive inoculations on non-selective media, rendering the strain incapable of metabolizing chlorinated benzenes. Subsequent cloning and deletion experiments in <em>Escherichia coli, Pseudomonas putida,</em> and <em>Alcaligenes eutrophus</em> showed that two regions on plasmid pP51 were responsible for chlorobenzene metabolism. Expression studies in <em>E. coli</em> revealed that a 5-kb region encoded the activity to convert 1,2,4-TCB and 1,2-DCB to 3,4,6-trichlorocatechol and 3,4-dichlorocatechol, respectively. This activity was determined using whole cell incubations, and in analogy with other described catabolic pathways it was proposed that the activity was caused by a chlorobenzene dioxygenase multienzyme complex and a dehydrogenase (encoded by <em>tcbA</em> and <em>tcbB,</em> respectively). Separated from the chlorobenzene dioxygenase gene cluster by approximately 6 kb a region was located which contained the genes for the conversion of chlorocatechols. Different DNA fragments of this region of pP51 were cloned in expression vectors in <em>E. coli, P. putida</em> and <em>A. eutrophus.</em> Both <em>P.</em><em>putida</em> KT2442 and <em>A. eutrophus</em> JMP222 could be complemented for growth on 3-chlorobenzoate by a 13-kb fragment of pP51, which indicated that a functional pathway for degradation of chlorocatechols was encoded on this fragment. Enzyme activity measurements and transformation reactions with 3,4-dichlorocatechol in cell extracts of <em>E. coli</em> harboring cloned pP51 DNA fragments showed the activity of three enzymes, chlorocatechol 1,2-dioxygenase (catechol 1,2-dioxygenase II), chloromuconate cycloisomerase (cycloisomerase II), and dienelactone hydrolase II. The genes encoding these activities were designated <em>tcbC, tcbD,</em> and <em>tcbE,</em> respectively, and their deduced gene order was found to be <em>tcbC-tcbD- tcbE.</em> It was thus proposed that 3,4-dichlorocatechol was converted via a chlorocatechol oxidative pathway (or modified <em>ortho</em> cleavage pathway), similar to that described in <em>Pseudomonas</em> sp. <em></em> strain B 13 and <em>A. eutrophus</em> JMP134 , leading finally to the formation of 5-chloromaleylacetate. The release of one chlorine atom from 3,4- dichlorocatechol was shown to take place spontaneously during lactonization in the cycloisomerization reaction.<p>The genes of the chlorocatechol oxidative pathway of strain P51 are organized in a single operon, comprising a region of 5.5 kb, which was fully sequenced and contained five large open reading frames (Chapter 4). The gene products of the different open reading frames were analyzed by subcloning appropriate pP51 DNA fragments in <em>E. coli</em> expression vectors. Expression studies confirmed the previously determined gene order and could attribute three open reading frames to the gene loci <em>tcbC, tcbD,</em> and <em>tcbE,</em> respectively. In between <em>tcbD</em> and <em>tcbE</em> an 1,022 bp open reading frame was present (ORF3), but we could not detect any protein encoded by this ORF. Immediately downstream of <em>tcbE</em> another ORF was found, designated <em>tcbF,</em> which encoded a 38 kDa protein. Until now, no clear function has been attributed for the <em>tcbF</em> gene product. The <em>tcbCDEF</em> genes and their encoded gene products showed high (50.6% - 75.7%) homology to two other chlorocatechol oxidative gene clusters, <em>clcABD</em> of <em>P.</em><em>putida</em> (pAC27) and <em>tfdCDEF</em> of <em>A. eutrophus</em> JMP134(pJP4). Furthermore, a homology of 22% and 43.9% was found of TcbC and TcbD to CatA and CatB, respectively, the catechol 1,2-dioxygenase and cycloisomerase of the β-ketoadipate pathway of <em>Acinetobacter calcoaceticus.</em> This suggests that the chlorocatechol oxidative pathway originated from other, more common, metabolic pathways. Despite the strong DNA and amino acid sequence homology of the genes and enzymes of the chlorocatechol oxidative pathways, the substrate range of the pathway enzymes from the three organisms differed subtly. This was demonstrated for the chlorocatechol 1,2- dioxygenases TcbC, ClcA, and TfdC. In contrast to ClcA and TfdC, which showed a high relative activity for 3,5-dichlorocatechol, TcbC exhibited a strong preference for 3,4- dichlorocatechol and a weak affinity for the 3,5-isomer. This suggested that the <em>tcb</em> -encoded pathway enzymes had become specialized for intermediates (i.e. 3,4- dichlorocatechol) which arise in the metabolism of the novel compound 1,2- dichlorobenzene. Different genetic mechanisms may cause the divergence of genes and allow a specialization of encoded proteins (see also Chapter 1). Recently it has been proposed that slippage of short sequence repetitive motifs and subsequent mismatch repair would be the major driving force for rapid evolutionary divergence, rather than single base-pair substitutions. Detailed DNA sequence comparisons between the chlorocatechol 1,2-dioxygenase genes <em>tcbC</em> , <em>clcA</em> , and <em>tfdC</em> gives evidence for slippage of short sequence repetitions in regions of strong divergence in amino acid sequence.<p>The transcription of the <em>tcbCDEF</em> operon <em></em> was found to be regulated by the gene product of <em>tcbR,</em> a gene located upstream of and divergently transcribed from the tcbC gene. The <em>tcbR gene</em> was characterized by DNA sequencing and expression studies in <em>E. coli</em> and appeared to encode a 32 kDa protein (Chapter 5). The activity of the <em>tcbR</em> gene was analyzed in <em>P.</em><em>putida</em> KT2442 harboring the cloned <em>tcbR</em> and <em>tcbCDEF</em> genes by determining the activity of the chlorocatechol 1,2-dioxygenase TcbC during growth on 3-chlorobenzoate. Strains of <em>P.</em><em>putida</em> KT2442, which carried a frame shift mutation in the <em>tcbR</em> gene, could no longer induce <em>tcbC</em> expression during growth on 3-chlorobenzoate, suggesting that TcbR functions as a positive regulator of <em>tcbC</em> expression. A region of 150-bp is separating <em>tcbR</em> and <em>tcbC,</em> the first gene of the <em>tcbCDEF</em> cluster, and contains the expression signals needed for the transcriptional activation of <em>tcbCDEF</em> by the <em>tcbR</em> gene product. The transcriptional start sites of <em>tcbR</em> and <em>tcbC</em> were determined by primer extension analysis and this showed that the two divergent promoter sequences of the genes overlap. Protein extracts of both <em>E. coli</em> overproducing TcbR and of <em>Pseudomonas</em> sp. <em></em> strain P51 showed specific DNA binding to this 150-bp region. TcbR probably regulates <em>tcbCDEF</em> expression and autoregulates its own expression, by binding the DNA region containing the promoters of <em>tcbC</em> and <em>tcbR.</em> It is likely that an inducer molecule interacts with TcbR, which may cause alterations or partially unwinding of the bound region and stimulation of RNA polymerase to start transcription of the <em>tcbCDEF</em> operon. Amino acid sequence comparisons indicated that TcbR is a member of the LysR family of transcriptional activator proteins and shares a high degree of homology with other activator proteins involved in regulating the catabolism of aromatic compounds, such as CatM, CatR and NahR. Detailed studies have recently been carried out to determine the precise interaction of TcbR with its operator region by DNasel footprinting. It would be interesting to see if in analogy with the specialization of TcbC, TcbR has diverged from a more common regulator protein such as CatM or CatR, and became specialized in recognizing chorinated inducer molecules.<p>DNA sequence analysis of the start of the chlorobenzene dioxygenase cluster revealed the presence of an insertion element, IS <em>1066</em> (Chapter 6). An almost exact copy of this element, IS <em>1067,</em> was discovered on the other side of this gene cluster, although oriented in an inverted position. Thus, the complete genetic element formed by IS <em>1066,</em> the <em>tcbAB</em> gene cluster, and IS <em>1067,</em> resembled a composite bacterial transposon. The functionality of this transposon, which was designated Tn <em>5280</em> , was established by inserting a 12-kb <em>Hin</em> dIII <em></em> fragment <em></em> of pP51 containing Tn <em>5280</em> , marked with a kanamycin resistance gene in between the IS-elements, into the suicide donor plasmid pSUP202 followed by conjugal transfer to <em>P.</em><em>putida</em> KT2442. Analysis by DNA hybridization of transconjugants with acquired kanamycin resistance showed that Tn <em>5280</em> had transposed into the genome of this strain at random and in single copy. The insertion elements IS <em>1066</em> and IS <em>1067</em> were found to be highly homologous to a class of repetitive elements of <em>Bradyrhizobium japonicum</em> and <em>Agrobacterium rhizogenes,</em> and were distantly related to IS <em>630</em> of <em>Shigella sonnei.</em> The presence of the <em>tcbAB</em> genes on Tri <em>5280</em> suggested a mechanism by which a chlorobenzene dioxygenase gene cluster was mobilized as a gene module by the mediation of IS-elements. This gene module was then joined with the chlorocatechol gene cluster to form the novel chlorobenzene pathway.<p>To obtain more information on the distribution of chlorobenzene degradation genes in the environment, different methods were applied which were based on DNA- DNA hybridization with gene probes derived from chloroaromatic metabolism (Chapter 7). A number of bacterial strains which were isolated by selective enrichment from soil samples for growth on chloroaromatic compounds .was screened for the presence of catabolic plasmids. Hybridization of these plasmid-DNA's with DNA fragments of the <em>tcbAB</em> or <em>tcbCDEF</em> genes revealed a class of plasmids which were identical or homologous to plasmid pP51 of strain P51. In other experiments soil microorganisms were directly extracted from soil samples, plated on nonselective media and screened by DNA-DNA colony hybridization for the presence of catabolic genes with a set of probes for three chlorocatechol 1,2-dioxygenase genes <em>(tcbC,</em> clcA, and <em>tfdC).</em> Positively reacting colonies were obtained under selective conditions with a frequency of 1 to 5 per 2000, which indicated that in the soil samples microorganisms were present which contained DNA sequences homologous to the used probes. However, from additional screening and hybridization experiments of these positively reacting colonies it became clear that some of these were false positives. Furthermore, positive strains were lost easily during transfer from the original agar plates due to the heterogeneity in colony types of the different soil microorganisms. In a third method the variation of chlorocatechol 1,2-dioxygenase genes among soil microorganisms was analyzed by amplifying total DNA from soil samples in the polymerase chain reaction, which was primed with degenerate oligonucleotides derived for conserved regions in <em>tcbC,</em> clcA, and <em>tfdC.</em> Discrete amplified fragments were obtained in this manner, which were cloned and analyzed by hybridization and DNA sequencing. We found six different types of fragments which had the expected size, only one of which was related significantly to the chlorocatechol 1,2-dioxygenase (and in fact was identical to the <em>tcbC- type).</em> This indicated that it was possible to detect and isolate chlorocatechol 1,2-dioxygenase sequences from soil DNA although the selectivity of the amplification reaction was relatively low.<p>In this study, we have entered a field of microbial research which will have continuing evolutionary and environmental interest. A detailed genetic characterization of bacteria which adapted to use xenobiotic compounds as novel growth and energy subsrates, suggested different mechanisms by which novel metabolic pathways evolve in bacteria. Our results presented evidence for i) a specialization of enzyme systems and ii) an exchange and combination of pre-existing gene modules. Still we do not know what the capacity of microorganisms present in the natural environment is to adapt rapidly to metabolize xenobiotic substrates, nor do we know how and which environmental factors influence genetic adaptation. Astonished by the diversity of genetic mechanisms displayed in bacteria which govern evolutionary change, we shouldn't be surprised to find mechanisms which direct and regulate genetic adaptation in response to changing environmental conditions.
Identification of a novel composite transposable element, Tn 5280, carrying chlorobenzene dioxygenase genes of Pseudomonas sp. strain P51.
Meer, J.R. van der; Zehnder, A.J.B. ; Vos, W.M. de - \ 1991
Journal of Bacteriology 173 (1991). - ISSN 0021-9193 - p. 7077 - 7083.
pseudomonas - micro-organismen - genetica - heritability - derivaten - benzeen - chloride - hexachloorbenzeen - adaptatie - microorganisms - genetics - derivatives - benzene - hexachlorobenzene - adaptation
|Characterization of the Pseudomonas sp. Strain P51 Gene tcbR, a LysR-type transcriptional activator of the tcb CDEF chlorocatechol oxidate operon, and analysis of the regulatory region.
Meer, J.R. van der; Frijters, A.C.J. ; Leveau, J.H.J. ; Eggen, R.I.L. ; Zehnder, A.J.B. ; Vos, W.M. de - \ 1991
Journal of Bacteriology 173 (1991). - ISSN 0021-9193 - p. 3700 - 3708.
benzeen - chloride - derivaten - genetische code - genetica - heritability - hexachloorbenzeen - micro-organismen - moleculaire biologie - pseudomonas - benzene - derivatives - genetic code - genetics - hexachlorobenzene - microorganisms - molecular biology
Sequence analysis of the Pseudomonas sp. strain P51 tcb gene cluster, which encodes metabolism of chlorinated catechols: evidence for specialization of catechol 1,2-dioxygenases for chlorinated substrates.
Meer, J.R. van der; Eggen, R.I.L. ; Zehnder, A.J.B. ; Vos, W.M. de - \ 1991
Journal of Bacteriology 173 (1991). - ISSN 0021-9193 - p. 2425 - 2434.
benzeen - chloride - derivaten - genetische code - genetica - heritability - hexachloorbenzeen - micro-organismen - moleculaire biologie - pseudomonas - benzene - derivatives - genetic code - genetics - hexachlorobenzene - microorganisms - molecular biology
Bacterial formation of hydroxylated aromatic compounds
Tweel, W.J.J. van den - \ 1988
Agricultural University. Promotor(en): J.A.M. de Bont, co-promotor(en): J. Tramper. - S.l. : van den Tweel - 197
benzeen - biochemie - biotechnologie - chemische industrie - derivaten - microbiële afbraak - organische verbindingen - synthese - chemische verbindingen - benzene - biochemistry - biotechnology - chemical industry - derivatives - microbial degradation - organic compounds - synthesis - chemical compounds
<p>As stated in the introduction of this thesis, hydroxylated aromatic compounds in general are of great importance for various industries as for instance pharmaceutical, agrochemical and petrochemical industries. Since these compounds can not be isolated in sufficient amounts from natural resources, they have to be synthesized. Chemical synthesis of hydroxylated aromatics is often a difficult task. Direct hydroxylation methods can only be achieved under extreme conditions, while indirect methods often are laborious multi-step processes. Biotechnological formation methods for hydroxylated aromatic compounds are a promissing alternative to the cumbersome organic chemical endeavours. The bioformation of hydroxylated aromatics in principle can be accomplished in four different ways: along biosynthetic routes, by means of direct hydroxylation methods, by replacement of substituents by hydroxyl groups, and by addition and/or modification reactions of side-chains. This research was done to investigate the potential of bacteria or enzymes thereof to form hydroxylated aromatics.<p>To obtain a microorganism which hydroxylates D-phenyIglycine regio- and stereospecifically yielding D-4-hydroxyphenyIglycine, various bacteria were isolated on D-phenyIglycine as sole carbon and energy source. Unfortunately, however, none of the isolates was able to hydroxylate phenylglycine (chapters 1 and 2). Experiments with whole cells and cell extracts showed that the side chain was modified before hydroxylation of the aromatic ring occurred. One of the isolates, <em>Pseudomonas putida</em> LW-4, was also able to grow on D-4-hydroxyphenyIglycine and it was shown that this compound was initially degraded by means of an enantioselective transaminase. Preliminary experiments with partially purified extracts have demonstrated that this reversible enzyme can be used to form D-4-hydroxyphenylglycine from 4-hydroxyphenylglyoxylate (chapter 4). To investigate D-4-hydroxyphenylglycine degradation in general, also other bacteria were isolated on D-4-hydroxyphenylglycine as sole carbon and energy source. One of these isolates, <em>Pseudomonas putida</em> MW27, possessed a D-selective as well as a L-selective 4-hydroxyphenylglycine transaminase (chapter 5). Evidently some microorganisms transaminate both enantiomers of 4-hydroxyphenylglycine and thus are less suitable for the formation of D-4-hydroxyphenylglycine by means of a trans amination.<p>To apply bacteria or enzymes thereof for the hydroxylation of phenylacetate and/or certain hydroxyphenylacetates a thorough knowledge concerning the bacterial metabolism of these compounds is needed. In chapter 6 the degradation of 4-hydroxyphenylacetate by a <em>Xanthobacter</em> species is described and it is shown that this strain can convert 4-hydroxyphenylacetate to 2,5-dihydroxyphenylacetate (homogentisate). To accomplish a formation of homogentisate by whole cells, further degradation of homogentisate had to be blocked by metalchelators. In chapter 7 the degradation of DL-phenylhydracrylic acid and metabolites thereof, by a <em>Flavobacterium</em> species is described. In the presence of dipyridyl these cells converted both 3- and 4-hydroxyphenylacetate to homogentisate. As stated in chapter 7, the internal regeneration of reduction equivalents by using starting compounds which are more reduced than the compound to be hydroxylated, might be an interesting alternative to the simple addition of cosubstrates.<p>The bioformation of cis- ,2-dihydroxycyclohexa-3,5-diene (cis-benzeneglycol) from benzene illustrates the potential of biotransformations. The chemical synthesis of cis-benzeneglycol consists of several steps with a very low yield, whereas the biological formation is a one step process with a high yield. Continuous bioformation of cis-benzeneglycol from benzene by mutant cells growing on succinate under nitrogen-limited conditions in a chemostat, was easily achieved (chapter 8). In order to predict the cis-benzeneglycol concentration at various times, a mathematical model was developed that fitted rather well for both benzene-transport-limited and kinetically limited production conditions. This continuous process, however, resulted in two products: cis-benzeneglycol and cells. In order to make the continuous process economically more attractive, it is necessary to reuse the produced cells. Another problem encountered during the bioproduction of cis-benzeneglycol was the toxicity of benzene; a low benzene concentration was a prerequisite for good performance of the bioconversion process. Incubation experiments with the cis-benzeneglycol-producing mutant showed that hexadecane is a suitable solvent to circumvent benzene toxicity (chapter 9). Moreover, the addition of hexadecane did not significantly effect the rate of cis-benzeneglycol formation.<p>Chapters 10, 11 and 12 deal with the bioformation of 4-hydroxybenzoate from various 4-halobenzoates. Bioformation of 4-hydroxybenzoate was only achieved when whole cells were incubated with the specified 4-halobenzoates under conditions of low and controlled oxygen concentrations. Surprisingly no formation of 4- hydroxybenzoate occurred under anaerobic conditions, this in spite of the fact that such dehalogenases have been demonstrated to be hydrolytic. 4-Hydroxybenzoate was also formed from 2,4-dichlorobenzoate. This latter compound was initially reductively dechlorinated to 4-chlorobenzoate which in turn was converted to 4-hydroxybenzoate (chapter 11). In order to study the feasibility of continuous bioproduction of hydroxyaromatics from haloaromatics, the bioconversion of 4-chlorobenzoate to 4-hydroxybenzoate by cells immobilized in carrageenan was used as a model system. At air saturation the rate of dechlorination was rapidly limited by internal oxygen transport. However, high oxygen concentration resulted in maximal 4-chlorobenzoate dehalogenation, while 4-hydroxybenzoate formation under these conditions was negligible. Consequently, the oxygen concentration has to be strictly controlled to obtain a good production of 4-hydroxybenzoate at an acceptable rate.
Adsorptie van de organische microverontreinigingen benzeen, tolueen en xyleen in eerdgrond : het effect van microbiologische activiteit op adsorptie
Bransen, F. - \ 1987
Wageningen : ICW (Nota / Instituut voor Cultuurtechniek en Waterhuishouding 1779) - 34
absorptie - adsorptie - benzeen - tolueen - xyleen - microbiologie - absorption - adsorption - benzene - toluene - xylene - microbiology