Little is known about the capacity of terrestrial invertebrates to transform organic soil pollutants such as polycyclic aromatic hydrocarbons (PAHs). Studies were designed to characterize microsomal mixed function oxygenase and accompanying conjugation enzymes from the hepatopancreas of the terrestrial isopods Porcellio scaber and Oniscus asellus using pyrene and 1-hydroxypyrene as model substrates. The hydroxylation of pyrene and the formation of pyreneglucoside and pyrenesulfate appeared to be sensitive measures for the activity of cytochrome P450 aryl hydrocarbon hydroxylase (AHH), uridinediphosphateglucosyltransferase (UDPGT), and aryl sulfotransferase (ST), respectively. Treatment with the antibiotic riphampicine demonstrated that the enzyme activities originate from the animals themselves and not from symbiotic microflora present in the hepatopancreas and the gut. In both species, ST has a very high affinity for 1-hydroxypyrene with Km values two orders of magnitude lower than that of UDPGT. The Vmax values of UDPGT, however, are 10- to 20-fold higher than that of ST. Taking the P450 activities into consideration, both species are expected to transform PAHs in an equally effective way. When the isopods were fed with food containing benz[a]pyrene and 3-methyl-cholanthrene, none of the enzyme activities appeared to be inducible except for a small enhancement of UDPGT in O. asellus. Our findings indicate that terrestrial isopods have a high, noninducible capacity for biotransformation of PAHs and that the sulfate conjugation pathway is as important as the carbohydrate conjugation pathway. This conclusion is consistent with the low body residues of parent PAHs found in the field.
Little is known about the capacity of terrestrial invertebrates to transform organic soil pollutants such as polycyclic aromatic hydrocarbons (PAHs). Studies were designed to characterize microsomal mixed function oxygenase and accompanying conjugation enzymes from the hepatopancreas of the terrestrial isopods Porcellio scaber and Oniscus asellus using pyrene and 1-hydroxypyrene as model substrates. The hydroxylation of pyrene and the formation of pyreneglucoside and pyrenesulfate appeared to be sensitive measures for the activity of cytochrome P450 aryl hydrocarbon hydroxylase (AHH), uridinediphosphateglucosyltransferase (UDPGT), and aryl sulfotransferase (ST), respectively. Treatment with the antibiotic riphampicine demonstrated that the enzyme activities originate from the animals themselves and not from symbiotic microflora present in the hepatopancreas and the gut. In both species, ST has a very high affinity for 1-hydroxypyrene with Km values two orders of magnitude lower than that of UDPGT. The Vmax values of UDPGT, however, are 10- to 20-fold higher than that of ST. Taking the P450 activities into consideration, both species are expected to transform PAHs in an equally effective way. When the isopods were fed with food containing benz[a]pyrene and 3-methyl-cholanthrene, none of the enzyme activities appeared to be inducible except for a small enhancement of UDPGT in O. asellus. Our findings indicate that terrestrial isopods have a high, noninducible capacity for biotransformation of PAHs and that the sulfate conjugation pathway is as important as the carbohydrate conjugation pathway. This conclusion is consistent with the low body residues of parent PAHs found in the field.
In the present study, the validity of using a cocktail screening method in combination with a chemometrical data mining approach to evaluate metabolic activity and diversity of drug-metabolizing bacterial Cytochrome P450 (CYP) BM3 mutants was investigated. In addition, the concept of utilizing an in-house-developed library of CYP BM3 mutants as a unique biocatalytic synthetic tool to support medicinal chemistry was evaluated. Metabolic efficiency of the mutant library towards a selection of CYP model substrates, being amitriptyline (AMI), buspirone (BUS), coumarine (COU), dextromethorphan (DEX), diclofenac (DIC) and norethisterone (NET), was investigated. First, metabolic activity of a selection of CYP BM3 mutants was screened against AMI and BUS. Subsequently, for a single CYP BM3 mutant, the effect of co-administration of multiple drugs on the metabolic activity and diversity towards AMI and BUS was investigated. Finally, a cocktail of AMI, BUS, COU, DEX, DIC and NET was screened against the whole in-house CYP BM3 library. Different validated quantitative and qualitative (U)HPLC-MS/MS-based analytical methods were applied to screen for substrate depletion and targeted product formation, followed by a more in-depth screen for metabolic diversity. A chemometrical approach was used to mine all data to search for unique metabolic properties of the mutants and allow classification of the mutants. The latter would open the possibility of obtaining a more in-depth mechanistic understanding of the metabolites. The presented method is the first MS-based method to screen CYP BM3 mutant libraries for diversity in combination with a chemometrical approach to interpret results and visualize differences between the tested mutants.Graphical abstractGeneral worklfow in screening mutant enzyme libraries for catalytic efficiency and diversityElectronic supplementary materialThe online version of this article (doi:10.1007/s00216-015-9241-x) contains supplementary material, which is available to authorized users.
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