Biomarkers of Flutamide-Bioactivation and Oxidative Stress In Vitro and In Vivo.

The nonsteroidal androgen-receptor antagonist flutamide is associated with hepatic injury. Oxidative stress and reactive metabolite formation are considered contributing factors to liver toxicity. Here we have used flutamide as a model drug to study the generation of reactive drug metabolites that undergo redox cycling to induce oxidative stress (OS) in vitro and in vivo. Lipid peroxidation (LPO) markers, as well as genes regulated by the redox-sensitive Nrf2 pathway, have been identified as surrogates for the characterization of OS. These markers and metabolism biomarkers for drug bioactivation have been investigated to characterize drug-induced hepatic damage. Rat hepatocytes and in vivo studies showed that several LPO markers, namely the isoprostanes 15R-PD2, dihydro keto PE2, and iPF(2α)-VI, as well as hydroxynonenal mercapturic acid metabolites, had increased significantly by 24 hours after flutamide treatment from 4.9 to 15.3-fold in hepatocytes and from 2.6 to 31.0-fold in rat plasma. Induction of mRNA expression levels for Nrf2-regulated genes was evident as well, with heme oxygenase 1, glutathione-S-transferase π1 and NAD(P)H dehydrogenase showing a 3.6-, 4.1-, and 1.9-fold increase in hepatocytes and 5.6-, 7.5-, and 94.1-fold in rat liver. All effects were observed at drug concentrations that did not show overt liver toxicity. Addition of an in situ hydrogen peroxide-generating system to in vitro experiments demonstrated the formation of a reactive di-imine intermediate as the responsible metabolic pathway for the generation of OS. The dataset suggests that hepatic oxidative stress conditions can be mediated via metabolic activation and can be monitored with suitable biomarkers preceding the terminal damage.

Quantitative profiling of prostaglandins as oxidative stress biomarkers in vitro and in vivo by negative ion online solid phase extraction - Liquid chromatography-tandem mass spectrometry.

Free radical-mediated oxidation of arachidonic acid to prostanoids has been implicated in a variety of pathophysiological conditions such as oxidative stress. Here, we report on the development of a liquid chromatography-mass spectrometry method to measure several classes of prostaglandin derivatives based on regioisomer-specific mass transitions down to levels of 20 pg/ml applied to the measurement of prostaglandin biomarkers in primary hepatocytes. The quantitative profiling of prostaglandin derivatives in rat and human hepatocytes revealed the increase of several isomers on stress response. In addition to the well-established markers for oxidative stress such as 8-iso-prostaglandin F2α and the prostaglandin isomers PE2 and PD2, this method revealed a significant increase of 15R-prostaglandin D2 from 236.1 ± 138.0 pg/1E6 cells in untreated rat hepatocytes to 2001 ± 577.1 pg/1E6 cells on treatment with ferric NTA (an Fe(3+) chelate with nitrilotriacetic acid causing oxidative stress in vitro as well as in vivo). Like 15R-prostaglandin D2, an unassigned isomer that revealed a more significant increase than commonly analyzed prostaglandin derivatives was identified. Mass spectrometric detection on a high-resolution instrument enabled high-quality quantitative analysis of analytes in plasma levels from rat experiments, where increased concentrations up to 23-fold change treatment with Fe(III)NTA were observed.

Software-aided cytochrome P450 reaction phenotyping and kinetic analysis in early drug discovery.

RATIONALE: Cytochrome P450 (CYP450) reaction phenotyping (CRP) and kinetic studies are essential in early drug discovery to determine which metabolic enzymes react with new drug entities. A new semi-automated computer-assisted workflow for CRP is introduced in this work. This workflow provides not only information regarding parent disappearance, but also metabolite identification and relative metabolite formation rates for kinetic analysis. METHODS: Time-course experiments based on incubating six probe substrates (dextromethorphan, imipramine, buspirone, midazolam, ethoxyresorufin and diclofenac) with recombinant human enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) and human liver microsomes (HLM) were performed. Liquid chromatography/high-resolution mass spectrometry (LC/HRMS) analysis was conducted with an internal standard to obtain high-resolution full-scan and MS/MS data. Data were analyzed using Mass-MetaSite software. A server application (WebMetabase) was used for data visualization and review. RESULTS: CRP experiments were performed, and the data were analyzed using a software-aided approach. This automated-evaluation approach led to (1) the detection of the CYP450 enzymes responsible for both substrate depletion and metabolite formation, (2) the identification of specific biotransformations, (3) the elucidation of metabolite structures based on MS/MS fragment analysis, and (4) the determination of the initial relative formation rates of major metabolites by CYP450 enzymes. CONCLUSIONS: This largely automated workflow enabled the efficient analysis of HRMS data, allowing rapid evaluation of the involvement of the main CYP450 enzymes in the metabolism of new molecules during drug discovery.

Application of lipid peroxidation products as biomarkers for flutamide-induced oxidative stress in vitro.

The early identification of hepatotoxicity is a fundamental goal of preclinical safety studies in drug discovery and early development. Sensitive biomarkers warrant the determination of potential underlying mechanisms that help characterizing a disruption of physiological conditions prior to cell death. This study shows the potential of different lipid peroxidation products, namely isoprostanes and hydroxynonenal (HNE) derivatives, to serve as early safety biomarkers of hepatotoxicity caused by oxidative stress as underlying mechanism. The hepatotoxic drug flutamide was used as model compound in primary hepatocytes. Incubation conditions were optimized by the addition of hydrogen peroxide generating substrates enhancing the cellular response upon oxidative stress. A time and dose dependent response of different isoprostanes and prostaglandins (15R-prostaglandin D2, prostaglandin E2, 13,14-dihydro-15-keto prostaglandin E2 and 5­iso prostaglandin F2α-VI) became manifest after 6 and 24h of treatment in 3.8- to 17.4-fold increased concentrations where no overt hepatocellular damage was observed. For HNE-mercapturic acid and its metabolite dihydroxynonene-mercapturic acid a similar response was evident with a 20- and 10-fold increase from control after 24 h of treatment, respectively. These data indicate that lipid peroxidation products as markers of reactive oxygen species are more sensitive than conventional cytotoxicity markers for an early detection of drug-induced liver injury.