Petri Reponen

Characterization of Diuron N-Demethylation by Mammalian Hepatic Microsomes and cDNA-Expressed Human Cytochrome P450 Enzymes

Diuron, a widely used herbicide and antifouling biocide, has been shown to persist in the environment and contaminate drinking water. It has been characterized as a “known/likely” human carcinogen. Whereas its environmental transformation and toxicity have been extensively examined, its metabolic characteristics in mammalian livers have not been reported. This study was designed to investigate diuron biotransformation and disposition because metabolic routes, metabolizing enzymes, interactions, interspecies differences, and interindividual variability are important for risk assessment purposes. The only metabolic pathway detected by liquid chromatography/mass spectometry in human liver homogenates and seven types of mammalian liver microsomes including human was demethylation at the terminal nitrogen atom. No other phase I or phase II metabolites were observed. The rank order of N-demethyldiuron formation in liver microsomes based on intrinsic clearance (Vmax/Km) was dog > monkey > rabbit > mouse > human > minipig > rat. All tested recombinant human cytochrome P450s (P450s) catalyzed diuron N-demethylation and the highest activities were possessed by CYP1A1, CYP1A2, CYP2C19, and CYP2D6. Relative contributions of human CYP1A2, CYP2C19, and CYP3A4 to hepatic diuron N-demethylation, based on average abundances of P450 enzymes in human liver microsomes, were approximately 60, 14, and 13%, respectively. Diuron inhibited relatively potently only CYP1A1/2 (IC50 4 μM). With human-derived and quantitative chemical-specific data, the uncertainty factors for animal to human differences and for human variability in toxicokinetics were within the range of the toxicokinetics default uncertainty/safety factors for chemical risk assessment.

Characterization of human cytochrome P450 induction by pesticides.

Pesticides are a large group of structurally diverse toxic chemicals. The toxicity may be modified by cytochrome P450 (CYP) enzyme activity. In the current study, we have investigated effects and mechanisms of 24 structurally varying pesticides on human CYP expression. Many pesticides were found to efficiently activate human pregnane X receptor (PXR) and/or constitutive androstane receptor (CAR). Out of the 24 compounds tested, 14 increased PXR- and 15 CAR-mediated luciferase activities at least 2-fold. While PXR was predominantly activated by pyrethroids, CAR was, in addition to pyrethroids, well activated by organophosphates and several carbamates. Induction of CYP mRNAs and catalytic activities was studied in the metabolically competent, human derived HepaRG cell line. CYP3A4 mRNA was induced most powerfully by pyrethroids; 50 μM cypermethrin increased CYP3A4 mRNA 35-fold. CYP2B6 was induced fairly equally by organophosphate, carbamate and pyrethroid compounds. Induction of CYP3A4 and CYP2B6 by these compound classes paralleled their effects on PXR and CAR. The urea herbicide diuron and the triazine herbicide atrazine induced CYP2B6 mRNA more than 10-fold, but did not activate CAR indicating that some pesticides may induce CYP2B6 via CAR-independent mechanisms. CYP catalyzed activities were induced much less than the corresponding mRNAs. At least in some cases, this is probably due to significant inhibition of CYP enzymes by the studied pesticides. Compared with human CAR activation and CYP2B6 expression, pesticides had much less effect on mouse CAR and CYP2B10 mRNA. Altogether, pesticides were found to be powerful human CYP inducers acting through both PXR and CAR.

In vitro metabolism and interactions of the fungicide metalaxyl in human liver preparations

In order to provide additional information for risk assessment of the fungicide metalaxyl, the main objectives were (1) to elucidate the interactions of metalaxyl with different human liver cytochrome P450 enzymes, (2) to tentitatively identify and (semi)quantify metabolites in vitro and (3) to identify human CYP enzymes responsible for metabolism. The mean inhibitory concentrations (IC(50)) for 7-pentoxyresorufin-O-dealkylation (CYP2B) and bupropion hydroxylation (2B6) were 48.9 and 41.7μM, respectively. The biotransformation reactions were hydroxylation, (di)demethylation and lactone formation. In human liver microsomes predominant metabolites were two hydroxymetalaxyl derivatives or atropisomers of one of the derivatives. On the basis of previous rat studies these could be N-(2-hydroxymethyl-6-methylphenyl)-N-(methoxyacetyl)alanine methyl ester and/or N-(2,6-dimethyl-5-hydroxyphenyl)-N-(methoxyacetyl)alanine methyl ester. The amounts of didemethylmetalaxyl N-(2,6-dimethylphenyl)-N-(hydroxyacetyl)alanine and lactone 4-(2,6-dimethylphenyl)-3-methylmorpholine-2,5-dione were higher in homogenates than microsomes. The carcinogenic 2,6-dimethylaniline was not detected. Among the nine major human CYPs, CYP3A4 was the only one responsible for metalaxyl hydroxylation, while CYP2B6 was the major isoform responsible for (di)demethylation and lactone formation.

Comparative metabolism of benfuracarb in in vitro mammalian hepatic microsomes model and its implications for chemical risk assessment.

In vitro metabolism of benfuracarb in liver microsomes from seven species was studied in order to quantitate species-specific metabolic profiles and enhance benfuracarb risk assessment by interspecies comparisons. Using LC-MS/MS, a total of seven phase-I-metabolites were detected from the extracted chromatograms and six of them were unequivocally identified. Benfuracarb was metabolized via two metabolic pathways, the sulfur oxidation pathway and nitrogen sulfur bond cleavage, yielding carbofuran, which metabolized further. Analysis of the metabolic profiles showed that benfuracarb was extensively metabolized with roughly similar profiles in different species in vitro. In vitro intrinsic clearance rates as well as calculated in vivo hepatic clearances indicated that all seven species metabolize benfuracarb via the carbofuran metabolic pathway more rapidly than the sulfoxidation pathway. The highest interspecies differences in hepatic clearance rate values were for mouse and rat liver microsomes compared to human, i.e. 4.8 and 4.1-fold higher, as illustrated by in vivo hepatic clearance of carbofuran. Overall, there are quantitative interspecies differences in the metabolic profiles and kinetics of benfuracarb biotransformation. These findings illustrate that in vitro studies of benfuracarb metabolite profiles and toxicokinetics are helpful for the proper selection and interpretation of animal models for toxicological evaluation and chemical risk assessment.

Metabolism of α-thujone in human hepatic preparations in vitro

This study aims to characterize the metabolism of α-thujone in human liver preparations in vitro and to identify the role of cytochrome P450 (CYP) and possibly other enzymes catalyzing α-thujone biotransformations. With a liquid chromatography–mass spectrometry (LC-MS) method developed for measuring α-thujone and four potential metabolites, it was demonstrated that human liver microsomes produced two major (7- and 4-hydroxy-thujone) and two minor (2-hydroxy-thujone and carvacrol) metabolites. Glutathione and cysteine conjugates were detected in human liver homogenates, but not quantified. No glucuronide or sulphate conjugates were detected. Major hydroxylations accounted for more than 90% of the primary microsomal metabolism of α-thujone. Screening of α-thujone metabolism with CYP recombinant enzymes indicated that CYP2A6 was principally responsible for the major 7- and 4-hydroxylation reactions, although CYP3A4 and CYP2B6 participated to a lesser extent and CYP3A4 and CYP2B6 catalyzed minor 2-hydroxylation. Based on the intrinsic efficiencies of different recombinant CYP enzymes and average abundances of these enzymes in human liver microsomes, CYP2A6 was calculated to be the most active enzyme in human liver microsomes, responsible for 70–80% of the metabolism on average. Inhibition screening indicated that α-thujone inhibited both CYP2A6 and CYP2B6, with 50% inhibitory concentration values of 15.4 and 17.5 µM, respectively.

Genetically Modified Caco-2 Cells With Improved Cytochrome P450 Metabolic Capacity

The human intestinal Caco-2 cell line has been extensively used as a model of small intestinal absorption but it lacks expression and function of cytochrome P450 enzymes, particularly CYP3A4 and CYP2C9, which are normally expressed in the intestinal epithelium. In order to increase the expression and activity of CYP isozymes in these cells, we created 2 novel Caco-2 sublines expressing chimeric constitutive androstane or pregnane X receptors and characterized these cells for their metabolic and absorption properties. In spite of elevated mRNA expression of transporters and differentiation markers, the permeation properties of the modified cell lines did not significantly differ from those of the wild-type cells. In contrast, the metabolic activity was increased beyond the currently used models. Specifically, CYP3A4 activity was increased up to 20-fold as compared to vitamin D treated wild-type Caco-2 cells.

Overview of the metabolism and interactions of pesticides in hepatic in vitro systems

Public concern about the impact of xenobiotics, including pesticides, on human health is greater than ever before. Pesticides constitute a potential risk to humans who are exposed to them directly and indirectly. Accordingly, the health risk assessment of pesticides is of utmost importance for the protection of human health. Risk assessment needs reliable scientific information, and one source of information is the characterisation of metabolic factors and toxicokinetics. Indeed, quantitative toxicokinetic data in humans are needed for human risk assessment to make reliable comparisons between individuals or between species. Because humans cannot be used as study subjects except in occasional circumstances, we have to rely upon in vitro experiments, human-derived techniques and in vitro–in vivo predictions. These methods combined with the advantage of novel analytical techniques (LC/TOF-MS; LC/MS-MS and LC-NMR) for metabolite identification and quantification enable the development of quantitative chemical-specific assessment factors. Here, we will briefly describe the in vitro techniques used to study in vitro metabolism and interactions, summarise the metabolic and kinetic properties of diuron as an herbicide and demonstrate how to apply current techniques to obtain relevant data for risk assessment.

Do Cytochrome P450 Enzymes Contribute to the Metabolism of Xenobiotics in Human

The cytochromes P450 (CYP) comprise a large multigene family of hemethiolate proteins which are of considerable importance in the metabolism of xenobiotics and endobiotics. CYP enzymes in humans as well as in other species have been intensively studied during recent years (Pelkonen et al., 2008; Turpeinen et al., 2007). It is possible to characterize metabolic reactions and routes, metabolic interactions, and to assign which CYP is involved in the metabolism of a certain xenobiotic by different in vitro approaches (Pelkonen et al., 2005; Pelkonen & Raunio, 2005; Hodgson and Rose, 2007a). Risk assessment needs reliable scientific information and one source of information is the characterization of the metabolic fate and toxicokinetics of compounds. Toxicokinetics refers to the movement of a xenobiotic into, through, and out of the body and is divided into several processes including absorption, distribution, metabolism, and excretion (ADME). Metabolism is one of the most important factors that can affect the overall toxic profile of a pesticide. During metabolism, the chemical is first biotransformed by phase I enzymes, usually by the cytochrome P450 (CYP) enzyme system, and then conjugated to a more soluble and excretable form by phase II conjugating enzyme systems (Guengerich & Shimada, 1991). In general, these enzymatic reactions are beneficial in that they help eliminate foreign compounds. Sometimes, however, these enzymes transform an otherwise harmless substance into a reactive form – a phenomenon known as metabolic activation (Guengerich & Shimada, 1991).Exposure to pesticides is a global challenge to risk assessment (Alavanja et al., 2004; Maroni et al., 2006). On a world-wide basis, acute pesticide poisoning is an important cause of morbidity and mortality. In an extrapolation, WHO/UNEP estimated that more than 3 million people were hospitalized for pesticide poisoning every year and that 220 000 died; it particularly noted that two-thirds of hospitalizations and the majority of deaths were attributable to intentional self-poisoning rather than to occupational or accidental poisoning (Konradsen et al., 2005; WHO/UNEP, 1990). Humans are inevitably exposed to pesticides in a variety of ways: at different dose levels and for varying periods of time (Boobis et al., 2008; Ellenhorns et al., 1997).