Khaled Abass

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.

Thujone and thujone-containing herbal medicinal and botanical products: Toxicological assessment

Thujone, a major component of the notoriously famous absinthe drink, is neurotoxic, although the current view rather downgrades its risk to humans. In animal studies, thujone inhibits the gamma-aminobutyric acid A (GABA(A)) receptor causing excitation and convulsions in a dose-dependent manner, although there are uncertainties about the doses required in humans. Toxicity of thujone has been extensively studied. Neurotoxicity is the principal toxic outcome in acute and chronic studies. There is some equivocal evidence of carcinogenicity in rats. Metabolism of thujone has been elucidated both in vitro and in vivo in several species and in vitro in human liver preparations. CYP2A6 is the principal metabolic enzyme, followed by CYP3A4 and, to a lesser extent, CYP2B6. CYP-associated metabolism may give rise to some potential pharmacogenetic and metabolic interaction consequences. Although the data base for determining exposure limits is of variable usefulness, the best estimates for allowable daily intakes via herbal preparations and diet are of the order of 3-7 mg/day. There are still important gaps in the knowledge required to assess thujone toxicity, the most important ones being human dose-concentration-effect relationships including the elucidation of bioavailability, and the actual toxicological consequences of potential pharmacogenetic variations and environmental factors.

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.

Rapid screening of commercially available herbal products for the inhibition of major human hepatic cytochrome P450 enzymes using the N-in-one cocktail

Self-administration of complementary products concurrently with conventional medication is increasingly common. The potential for cytochrome P450 (CYP) inhibition requires investigation. The N-in-one assay with ten probe substrates for nine CYPs was used with human liver microsomes to investigate ten products. CYP inhibition was measured in a single liquid chromatography-tandem mass spectrometry (LC/MS-MS) analysis. Estimated IC50-values were determined for the extracts that produced significant inhibition (less than 100 μg ml−1). Inhibition of CYP2C19 by dong quai (IC50 = 13.7–14.3 μg ml−1 for the methanolic extract) and CYP2D6 by goldenseal (IC50 = 6.7 and 6.3 μg ml−1 for the aqueous and methanolic extracts, respectively), are of particular concern as the potential for adverse interactions is high. The inhibition of CYP2C8 by horsetail (IC50 = 93 μg ml−1 for the aqueous extract) requires further investigation, as the potential for concurrent use with products that require CYP2C8 for metabolism is significant. CYP3A4 inhibition varied depending on the probe reaction being monitored. The earlier reported findings of inhibition by black cohosh, goldenseal and gotu kola were confirmed. The present work has shown that the N-in-one cocktail is a rapid and reliable method that can be used as an initial screen to help prioritize products that require more detailed investigations and it can also be applied to monitor product variability.

An evaluation of the cytochrome P450 inhibition potential of selected pesticides in human hepatic microsomes

The goal of this work was to study the ability of 18 pesticides to inhibit selective model activities for all major xenobiotic-metabolizing enzymes, namely CYP1A1/2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4. Generally organophosphorus insecticides were the most potent and extensive inhibitors, especially towards CYP1A1/2 (IC50 values of chlorpyrifos, fenitrothion and profenofos ∼3 μ M), CYP2B6 (IC50 values of chlorpyrifos and fenitrothion 2.5 μ M), CYP2C8 (fenitrothion 4.3 μ M), CYP2C9 (fenitrothion and malathion 4.8 and 2.5 μ M, respectively), CYP2D6 (chlorpyrifos and phenthoate ∼ 3 μ M) and CYP3A4 (chlorpyrifos, fenitrothion and phenthoate 3–4 μ M). Otherwise there were quite considerable differences in potency and extent of inhibition between different organophosphates. Pyrethroids were in general very weak or inactive. Deltamethrin and fenvalerate were potent inhibitors of CYP2D6 (IC50 values of ∼ 3 μ M) while lambda-cyhalothrin potently inhibited both CYP2D6 and CYP3A4-mediated activities (IC50s about 3–4 μ M). Some pesticides caused relatively potent inhibitions sporadically (carbendazim, CYP2D6, IC50 = 12 μ M; atrazine, CYP3A4, IC50 = 2.8 μ M; glyphosate, CYP2C9, IC50 = 3.7 μ M; hexaflumuron, IC50 = 6.0 μ M). With the exceptions of alpha-cypermethrin, cypermethrin, isoproturon, carbaryl and abamectin, most pesticides inhibited relatively potently at least one CYP-selective activity, which may have relevance for potential interactions in occupational exposures and for further studies on the CYP-associated metabolism of respective pesticides.

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 Pesticides by Human Cytochrome P450 Enzymes In Vitro – A Survey

Cytochrome P450 enzymes (CYPs) are active in the metabolism of wide variety of xenobiotics. The investigation of the contributions of human CYPs in pesticides metabolism, especially insecticides, is still growing. One of the background tools to facilitate this task is by sorting the contribution of each human CYP isoform in the metabolism of pesticides. This paper attempts to provide a comprehensive literature survey on the role of human hepatic CYPs such as human CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5 and CYP3A7 in pesticides biotransformation in vitro as well as to sort the reactions mediated. Based on relevant publications identified by searching databases from 1995 through 2011, more than 400 metabolic reactions were reported to be mediated at least in part by human CYPs in vitro. Some information on older papers was obtained from previous literature surveys compiled by Hodgson 2001 & 2003. Finally, we give brief insight into potential modulations and consequences of human CYP genes – pesticides interactions.

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.

Estimation of health risk by using toxicokinetic modelling: A case study of polychlorinated biphenyl PCB153

To assess potential PCB153-associated human health effects and risks, it is necessary to model past exposure. PCB153 blood concentrations, obtained from the AMAP biomonitoring programme, in Inuit women covering the years 1994-2006 at Disko Bay, 1999-2005 at Nuuk, and 1992-2007 at Nunavik were used to extrapolate body burden and exposure to the whole lifespan of the population by the one-compartment toxicokinetic model. By using risk characterisation modelling, calculated Hazard Quotients were higher than 1 between the years 1955 and 1987 for the 90th population percentile and during 1956-1984 for the 50th population percentile. Cancer risk for overall exposure of PCB153 ranged from 4.6×10(-5) to 1.8×10(-6) for the 90th percentile and 3.6×10(-5) to 1.4×10(-10) for the 50th percentile between 1930 and 2049, when central estimates or upper-bound slope factors were applied. Cancer risk was below 1×10(-6) for the same time period when a lower slope factor was applied. Significant future research requirements to improve health risk characterisation include, among others, larger sample sizes, better analytical accuracy, fewer assumptions in exposure assessment, and consequently, a better choice of the toxicity benchmark used to develop the hazard quotient.