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.

Drug interactions with mitotane by induction of CYP3A4 metabolism in the clinical management of adrenocortical carcinoma.

Mitotane [1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,2-dichloroethane, (o,p'-DDD)] is the only drug approved for the treatment for adrenocortical carcinoma (ACC) and has also been used for various forms of glucocorticoid excess. Through still largely unknown mechanisms, mitotane inhibits adrenal steroid synthesis and adrenocortical cell proliferation. Mitotane increases hepatic metabolism of cortisol, and an increased replacement dose of glucocorticoids is standard of care during mitotane treatment.

Strategies in case of positive in vivo results in genotoxicity testing.

At the 2009 International Workshop on Genotoxicity Testing in Basel, an expert group gathered to provide guidance on suitable follow-up tests to describe risk when basic in vivo genotoxicity tests have yielded positive results. The working group agreed that non-linear dose-response curves occur in vivo with at least some DNA-reactive agents. Quantitative risk assessment in such cases requires the use of (1) adequate data, i.

Quantification of cortisol and 6 beta-hydroxycortisol in human urine by LC-MS/MS, and gender-specific evaluation of the metabolic ratio as biomarker of CYP3A activity.

Drug-drug and food-drug interactions are often due to an inhibition or induction of drug-metabolizing cytochrome P450 (CYP) enzymes and may result in non-response or adverse reactions. Hence, phenotypic biomarkers of CYP activity appear as useful tools for individualized pharmacotherapy. The metabolic ratio (MR) of the concentration of 6beta-hydroxycortisol (6beta-OHC) to cortisol (MR 6beta-OHC/cortisol) in human urine had been proposed as an endogenous marker for CYP3A activity.

The Viracept (nelfinavir)--ethyl methanesulfonate case: a threshold risk assessment for human exposure to a genotoxic drug contamination?

In May 2007, the F. Hoffmann-La Roche Company became aware of a contamination of Viracept (nelfinavir) tablets by the mutagenic DNA-ethylating agent ethyl methanesulfonate (EMS) as a result of a production incident. HIV-patients could have been exposed for 3 months to daily doses of up to 2.

Biological significance of DNA adducts: comparison of increments over background for various biomarkers of genotoxicity in L5178Y tk(+/-) mouse lymphoma cells treated with hydrogen peroxide and cumene hydroperoxide.

DNA is affected by background damage of the order of one lesion per one hundred thousand nucleotides, with depurination and oxidative damage accounting for a major part. This damage contributes to spontaneous mutation and cancer. DNA adducts can be measured with high sensitivity, with limits of detection lower than one adduct per one billion nucleotides.

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.

Dose-response relationship for the pharmacokinetic interaction of grapefruit juice with dextromethorphan investigated by human urinary metabolite profiles.

Grapefruit juice (GFJ) has been shown to affect the pharmacokinetics of a large number of drugs, essentially by inhibition of efflux transporters and CYP3A4 monooxygenase in the small intestine. The GFJ dose usually used in human studies was one glass single-strength (1x). Information on a respective dose-response relationship is not available.

Pivotal role for two electron reduction in 2,3-dimethoxy-1,4-naphthoquinone and 2-methyl-1,4-naphthoquinone metabolism and kinetics in vivo that prevents liver redox stress.

2,3-dimethoxy-1,4-naphthoquinone (CAS-RN 6959-96-3) (DMNQ) and 2-methyl-1,4-naphthoquinone (CAS-RN 58-27-5) (MNQ:menadione) are effective one electron redox cycling chemicals in vitro. In addition, in vitro MNQ forms a thioether conjugate with glutathione by nucleophilic attack at the third carbon. In contrast, here we demonstrate that in vivo the major metabolic route is directly to the dihydronaphthoquinone for both DMNQ and MNQ followed by conjugation to mono- and di-glucuronides and sulfate.

Tests for genotoxicity and mutagenicity of furan and its metabolite cis-2-butene-1,4-dial in L5178Y tk+/- mouse lymphoma cells.

Furan is found in various food items and is cytotoxic and carcinogenic in the liver of rats and mice. Metabolism of furan includes the formation of an unsaturated dialdehyde, cis-2-butene-1,4-dial (BDA). In view of the multifunctional electrophilic reactivity of BDA, adduct formation with protein and DNA may explain some of the toxic effects.

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.

Biomarkers of furan exposure by metabolic profiling of rat urine with liquid chromatography-tandem mass spectrometry and principal component analysis.

Furan has been found in a number of heated food items and is carcinogenic in the liver of rats and mice. Estimates of human exposure on the basis of concentrations measured in food are not reliable because of the volatility of furan. A biomarker approach is therefore indicated.

Biological significance of DNA adducts investigated by simultaneous analysis of different endpoints of genotoxicity in L5178Y mouse lymphoma cells treated with methyl methanesulfonate.

The biological significance of DNA adducts is under continuous discussion because analytical developments allow determination of adducts at ever lower levels. Central questions refer to the biological consequences of adducts and to the relationship between background DNA damage and exposure-related increments. These questions were addressed by measuring the two DNA adducts 7-methylguanine (7-mG) and O(6)-methyl-2'-deoxyguanosine (O(6)-mdGuo) by LC-MS/MS in parallel to two biological endpoints of genotoxicity (comet assay and in vitro micronucleus test), using large batches of L5178Y mouse lymphoma cells treated with methyl methanesulfonate (MMS).

Ochratoxin A: 13-week oral toxicity and cell proliferation in male F344/n rats.

Ochratoxin A (OTA) is nephrotoxic and a potent renal carcinogen. Male rats are most susceptible to OTA toxicity, and chronic administration of OTA (70 and 210 microg/kg bw) for 2 years has been shown to induce high incidences of adenomas and carcinomas arising from the straight segment of the proximal tubule epithelium. In contrast, treatment with a lower dose of 21 microg/kg bw did not result in increased tumor rates, suggesting a nonlinear dose response for renal tumor formation by OTA.

Comparison of lanosterol-14 alpha-demethylase (CYP51) of human and Candida albicans for inhibition by different antifungal azoles.

Inhibition of fungal lanosterol-14 alpha-demethylase (CYP51) is the working principle of the antifungal activity of azoles used in agriculture and medicine. Inhibition of human CYP51 may result in endocrine disruption since follicular fluid-meiosis activating steroid (FF-MAS), the direct product of lanosterol demethylation, is involved in the control of meiosis. To investigate the specificity of antifungal agents for the fungal enzyme, assays to determine inhibitory potencies of 13 agricultural fungicides and 6 antimycotic drugs were established.

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.

Increased brain levels of 4-hydroxy-2-nonenal glutathione conjugates in severe Alzheimer's disease.

In the last decade an important role for the progression of neuronal cell death in Alzheimer's disease (AD) has been ascribed to oxidative stress. trans-4-Hydroxy-2-nonenal, a product of lipid peroxidation, forms conjugates with a variety of nucleophilic groups such as thiols or amino moieties. Here we report for the first time the quantitation of glutathione conjugates of trans-4-hydroxy-2-nonenal (HNEGSH) in the human postmortem brain using the specific and very sensitive method of electrospray ionization triple quadrupole mass spectrometry (ESI-MS-MS).

Time-dependent effects of sodium arsenite on DNA breakage and apoptosis observed in the comet assay.

To assess genotoxic effects of sodium arsenite (NaAsO2) the single-cell gel electrophoresis (comet assay) had been conducted in various studies indicating genotoxicity. However, DNA fragmentation due to NaAsO2-induced apoptosis may constitute a bias in the interpretation of the results. Apoptotic cells can show typically large and diffuse comets, which are usually excluded during genotoxicity analysis.

Inhibition of human CYP19 by azoles used as antifungal agents and aromatase inhibitors, using a new LC-MS/MS method for the analysis of estradiol product formation.

Azoles are used as fungicides in agriculture or antifungal drugs in medicine. Their therapeutic activity is based on the inhibition of fungal lanosterol-14alpha-demethylase (CYP51). Azoles are also used for the treatment of estrogen-dependent diseases, e.

Simultaneous determination of O6-methyl-2'-deoxyguanosine, 8-oxo-7,8-dihydro-2'-deoxyguanosine, and 1,N6-etheno-2'-deoxyadenosine in DNA using on-line sample preparation by HPLC column switching coupled to ESI-MS/MS.

O(6)-Methyl-2'-deoxyguanosine (O(6)-mdGuo), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), and 1,N(6)-etheno-2'-deoxyadenosine (epsilondAdo) are promutagenic DNA lesions originating from both endogenous and exogenous agents and actions (methylation, hydroxylation, lipid peroxidation products). A highly sensitive quantitative method was developed to measure these DNA adducts simultaneously, using liquid chromatography tandem mass spectrometry with column switching. Deuterated O(6)-[(2)H(3)]mdGuo was synthesized and used as internal standard.

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").