CYP2C9 genotype vs. metabolic phenotype for individual drug dosing--a correlation analysis using flurbiprofen as probe drug.

Currently, genotyping of patients for polymorphic enzymes responsible for metabolic elimination is considered a possibility to adjust drug dose levels. For a patient to profit from this procedure, the interindividual differences in drug metabolism within one genotype should be smaller than those between different genotypes. We studied a large cohort of healthy young adults (283 subjects), correlating their CYP2C9 genotype to a simple phenotyping metric, using flurbiprofen as probe drug.

Nano-stenciled RGD-gold patterns that inhibit focal contact maturation induce lamellipodia formation in fibroblasts.

Cultured fibroblasts adhere to extracellular substrates by means of cell-matrix adhesions that are assembled in a hierarchical way, thereby gaining in protein complexity and size. Here we asked how restricting the size of cell-matrix adhesions affects cell morphology and behavior. Using a nanostencil technique, culture substrates were patterned with gold squares of a width and spacing between 250 nm and 2 µm.

Pericellular fibronectin is required for RhoA-dependent responses to cyclic strain in fibroblasts.

To test the hypothesis that the pericellular fibronectin matrix is involved in mechanotransduction, we compared the response of normal and fibronectin-deficient mouse fibroblasts to cyclic substrate strain. Normal fibroblasts seeded on vitronectin in fibronectin-depleted medium deposited their own fibronectin matrix. In cultures exposed to cyclic strain, RhoA was activated, actin-stress fibers became more prominent, MAL/MKL1 shuttled to the nucleus, and mRNA encoding tenascin-C was induced.

Identification of a fibronectin interaction site in the extracellular matrix protein ameloblastin.

Mammalian teeth are composed of hydroxyapatite crystals that are embedded in a rich extracellular matrix. This matrix is produced by only two cell types, the mesenchymal odontoblasts and the ectodermal ameloblasts. Ameloblasts secrete the enamel proteins amelogenin, ameloblastin, enamelin and amelotin.

Statistical model to estimate a threshold dose and its confidence limits for the analysis of sublinear dose-response relationships, exemplified for mutagenicity data.

Strongly sublinear dose-response relationships (slope increasing with dose) raise the question about a putative threshold dose below which no biologically relevant effect would be expected. A mathematical threshold with a break in the curve at the threshold dose is generally rejected for consequences of genotoxicity such as mutation, because proportionality between low dose and the rate of DNA-adduct formation is a reasonable hypothesis. In view of an increasing database for distinct deviation from linearity for mutagenicity, we offer a statistical model to analyze continuous response data and estimate a threshold dose together with its confidence limits, thereby taking data quality and degree of sublinearity into account.

From mechanotransduction to extracellular matrix gene expression in fibroblasts.

Tissue mechanics provide an important context for tissue growth, maintenance and function. On the level of organs, external mechanical forces largely influence the control of tissue homeostasis by endo- and paracrine factors. On the cellular level, it is well known that most normal cell types depend on physical interactions with their extracellular matrix in order to respond efficiently to growth factors.

Metabolite profiling in human urine by LC-MS/MS: method optimization and application for glucuronides from dextromethorphan metabolism.

Analysis of human urine for specific compounds or metabolites is an established method for biomonitoring occupational or environmental exposures. Modern liquid chromatography-tandem mass spectrometry is not limited to single compounds but can simultaneously analyze whole classes of urine constituents with both high sensitivity and specificity. Individual differences in the composition of urine are very large in humans, which raises a number of problems that are not encountered in animal experimentation.

Tenascin-C induction by cyclic strain requires integrin-linked kinase.

Induction of tenascin-C mRNA by cyclic strain in fibroblasts depends on RhoA and Rho dependent kinase (ROCK). Here we show that integrin-linked kinase (ILK) is required upstream of this pathway. In ILK-deficient fibroblasts, RhoA was not activated and tenascin-C mRNA remained low after cyclic strain; tenascin-C expression was unaffected by ROCK inhibition.

Metabolic profiling of glucuronides in human urine by LC-MS/MS and partial least-squares discriminant analysis for classification and prediction of gender.

Mass spectrometry (MS) is increasingly being used for metabolic profiling, but detection modes such as constant neutral loss or multiple reaction monitoring have not often been reported. These modes allow focusing on structurally related compounds, which could be advantageous for situations in which the trait under investigation is associated with a particular class of metabolites. In this study, we analyzed endogenous glucuronides excreted in human urine by monitoring characteristic transitions of putative steroid glucuronides by LC-MS/MS for discrimination of females from males.

Dose-incidence modeling: consequences of linking quantal measures of response to depletion of critical tissue targets.

In developing mechanistic PK-PD models, incidence of toxic responses in a population has to be described in relation to measures of biologically effective dose (BED). We have developed a simple dose-incidence model that links incidence with BED for compounds that cause toxicity by depleting critical cellular target molecules. The BED in this model was the proportion of target molecule adducted by the dose of toxic compound.

Dose-incidence relationships derived from superposition of distributions of individual susceptibility on mechanism-based dose responses for biological effects.

Dose-response relationships for incidence are based on quantal response measures. A defined effect is either present or not present in an individual. The dose-incidence curve therefore reflects differences in individual susceptibility (the "tolerance distribution").

Different types of combination effects for the induction of micronuclei in mouse lymphoma cells by binary mixtures of the genotoxic agents MMS, MNU, and genistein.

Distinction between dose addition and response addition for the analysis of the toxicity of mixtures may allow differentiation of the components regarding similar versus independent mode of action. For nonlinear dose responses for the components, curves of dose addition and response addition differ and embrace an "envelope of additivity." Synergistic or antagonistic interaction may then be postulated only if the mixture effect is outside this surface.

LC-MS/MS analysis of dextromethorphan metabolism in human saliva and urine to determine CYP2D6 phenotype and individual variability in N-demethylation and glucuronidation.

In order to establish a fast screening method for the determination of the CYP2D6 metabolic phenotype a sensitive LC-MS/MS assay to quantify dextromethorphan (DEX) and its O-demethylated metabolite dextrorphan (DOR) in human saliva was developed with limits of quantitation of 1 pmol/ml. Saliva was provided by 170 medical students 2h after oral ingestion of 30 mg (81 micromol) dextromethorphan hydrobromide. Individual ratios of the concentrations DEX/DOR (metabolic ratio, MR(DEX/DOR)) varied more than 25,000-fold (0.

Comparative assessment of the inhibition of recombinant human CYP19 (aromatase) by azoles used in agriculture and as drugs for humans.

Azoles (imidazoles and triazoles) are used as antifungal agents in agriculture and in medicine, and also for antiestrogen therapy, e.g., for breast cancer treatment.

Statistical procedures to test for linearity and estimate threshold doses for tumor induction with nonlinear dose-response relationships in bioassays for carcinogenicity.

Sublinear shapes of the dose-response curve in the low-dose range of toxicity testing are often postulated to be indicative of a no-effect threshold. We present statistical procedures to test sublinear dose responses in bioassays for carcinogenicity against the hypothesis of linearity and estimate a lower confidence limit for the dose at the postulated breakpoint. First, a control tumor incidence of 0 is assumed.