Circadian and chemotherapy-related changes in urinary modified nucleosides excretion in patients with metastatic colorectal cancer.

Urinary levels of modified nucleosides reflect nucleic acids turnover and can serve as non-invasive biomarkers for monitoring tumour circadian dynamics, and treatment responses in patients with metastatic colorectal cancer. In 39 patients, median overnight urinary excretion of LC-HRMS determinations of pseudouridine, was ~ tenfold as large as those of 1-methylguanosine, 1-methyladenosine, or 4-acetylcytidine, and ~ 100-fold as large as those of adenosine and cytidine. An increase in any nucleoside excretion after chemotherapy anticipated plasma carcinoembryonic antigen progression 1-2 months later and was associated with poor survival. Ten fractionated urines were collected over 2-days in 29 patients. The median value of the rhythm-adjusted mean of urinary nucleoside excretion varied from 64.3 for pseudouridine down to 0.61 for cytidine. The rhythm amplitudes relative to the 24-h mean of 6 nucleoside excretions were associated with rest duration, supporting a tight link between nucleosides turnover and the rest-activity rhythm. Moreover, the amplitude of the 1-methylguanosine rhythm was correlated with the rest-activity dichotomy index, a significant predictor of survival outcome in prior studies. In conclusion, urinary excretion dynamics of modified nucleosides appeared useful for the characterization of the circadian control of cellular proliferation and for tracking early responses to treatments in colorectal cancer patients.

Dynamical thermal dose models and dose time-profile effects.

Introduction: Models of dose-effect relationships seek systematic and predictive descriptions of how cell survival depends on the level and duration of the stressor. The CEM43 thermal dose model has been empirically derived more than thirty years ago and still serves as a benchmark for hyperthermia protocols despitethe advent of regulatory network models. Objective: In this paper, we propose and realize a simple experimental test to assess whether mechanistic models can prove more reliable indicators for some protocols. We define two time-asymmetric hyperthermia profiles, faster rise than decay or slower rise than decay, for which the CEM43 model predicts the same survival while a regulatory network model predicts significant differences. Materials: Experimental data (both control 37°C and hyperthermia assays) were collected from duplicate HeLa cell cultures. Cells were imaged before and 24, 48 and 72 h after the hyperthermia assay double-stained with fluorescein-5-isothiocyanate (FITC)-labeled annexin V and propidium iodide for detecting cell death. Results: Survival experiments of HeLa cells show that a fast temperature rise followed by a slow decay can be twice more lethal than the opposite, consistently with the prediction of the network model. Conclusions: Using a model reduction approach, we obtained a simple nonlinear dynamic equation that identifies the limited repair capacity as the main factor underlying the dose-asymmetry effect and that could be useful for refining thermal doses for dynamic protocols.

Biological monitoring of occupational exposure to di(2-ethylhexyl) phthalate: survey of workers exposed to plastisols.

OBJECTIVE: The aim of this study was to perform a biological monitoring survey of workers exposed to di(2-ethylhexyl)phthalate (DEHP) in a factory using polyvinyl chloride plastisols and to contribute additional occupational data of exposure particularly sparse in the industrial sectors where this plasticizer is used. METHOD: Three urinary metabolites of DEHP, mono(2-ethylhexyl)phthalate (MEHP), mono(5-carboxy-2-ethylpentyl)phthalate (MCEPP) and 2-ethylhexanoic acid (2-EHA) were quantified in five workers using a plastisol (containing 33% of DEHP) and in five unexposed workers (controls), during 5 days with pre- and post-shift sampling. Analyses were performed by high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) with on-line extraction. RESULTS: Median concentrations of pre- and post-shift urinary samples in the exposed workers (n = 25) were 16.1 and 55.9 microg/l for MEHP, 37.6 and 103.7 microg/l for MCEPP and 46.3 and 72.1 microg/l for 2-EHA, respectively. In the controls (n = 19), the corresponding values were 12.0 and 10.4 microg/l for MEHP, 38.1 and 11.4 microg/l for MCEPP and 31.9 and 46.0 microg/l for 2-EHA, respectively. There is a significant increase (Mann-Whitney U-test, P < 0.05) of post-shift excretion in the exposed workers versus unexposed controls and in post-shift versus pre-shift concentrations only in the exposed workers. CONCLUSION: MEHP and MCEPP are shown to be suitable biomarkers to assess DEHP exposure while 2-EHA, less specific but classified in the category 3 of the European Union (EU) reproductive toxicants, is also an interesting biomarker. There is clear evidence of occupational exposure of workers in this factory.

Quantification of small therapeutic proteins in plasma by liquid chromatography-tandem mass spectrometry: application to an elastase inhibitor EPI-hNE4.

LC/ESI-MS/MS is a promising alternative to immunoassays in improving the analysis of recombinant therapeutic proteins in biological fluids for toxicity and pharmacokinetics purposes. To assess the sensitivity and validation issues associated with this technique, we use here as a model EPI-hNE4, a 56-amino acid recombinant protein, and demonstrate that a method based on tandem mass spectrometry combined with liquid chromatography and electrospray interface can reach sensitivity similar to that of ELISA but without its potential cross-reactivity. For this purpose, a triple quadrupole mass spectrometer operating in positive ion and single reaction monitoring mode with transition, m/z 1040 --> 1224.5, was used for selective peak detection. Particular issues related to the internal standard, i.e., elution and ionization patterns similar to the protein without stable isotope labeling, and to analytical interference due to endogenous binding antibodies were addressed. A limit of quantification in human or monkey plasma of 5 ng/mL was reached with a sample volume of 100 microL, corresponding to 40 fmol injected into the HPLC column. Intra- and interassay precision and accuracy were below 15%. No matrix effect was detected.

Cytochrome P450-mediated oxidation of glucuronide derivatives: example of estradiol-17beta-glucuronide oxidation to 2-hydroxy-estradiol-17beta-glucuronide by CYP 2C8.

In the classical metabolic oxidation scheme, hydrophobic endogenous or xenobiotic compounds undergo phase I oxidation, generally catalyzed in the liver by cytochromes P450, followed by phase II conjugation reactions, in a way that allows much more polar metabolites to be expelled from the cell through active transport mechanisms. Cytochrome P450-mediated oxidation of steroid sulfate has been described, suggesting that oxidation of polar metabolites such as glucuronide derivatives of endogenous compounds can occur. As an example, we report here that hydroxyestradiol-17beta-glucuronide can be directly formed through oxidation of estradiol-17beta-glucuronide on the aromatic C2 position. This reaction is specifically catalyzed by CYP 2C8, which is more active in female than in male human liver microsomes. A thorough docking of the molecule within the CYP 2C8 crystal structure shows that the active site is large enough to handle a glucuronide conjugate. Moreover, the most energetically favored position of the bound ligand is fully consistent with the recently published structural determinants of substrate specificity of the CYP 2C8 active site. This is the first demonstration of cytochrome P450-mediated oxidation of a steroid glucuro-conjugate. Such oxidation of a glucuronide should be a general process since, in addition to estradiol and testosterone glucuronide, it has been observed for xenobiotic compounds, e.g., diclofenac or naproxen glucuronide.

Ontogenesis of CYP2C-dependent arachidonic acid metabolism in the human liver: relationship with sudden infant death syndrome.

A modification of the human monooxygenase system have been previously associated with the sudden infant death syndrome (SIDS): the hepatic CYP2C content was markedly enhanced and resulted from an activation of CYP2C gene transcription. To determine the possible consequence of the up-regulation of CYP2C in SIDS, we examined the metabolism of arachidonic acid (AA) an endogenous substrate of CYP2C involved in the physiologic regulation of vascular tone. The overall AA metabolism was extremely low during the fetal period and rose after birth to generate 14,15 epoxyeicosatrienoic acid (EET), 11,12 EET and the sum of 5,6 dihydroxyeicosatrienoic acid (diHETE)+omega/omega-1 hydroxy AA. In SIDS, the accumulation of CYP2C proteins was associated with a significant increase in the formation of 14,15 and 11,12 diHETE, which were shown to be supported by individually expressed CYP2C8 and 2C9 and HETE1 (presumably 15 HETE). This increase was markedly inhibited by addition of sulfaphenazole, a selective inhibitor of CYP2C9. So, we propose that the higher CYP2C content in SIDS stimulates the production of EETs and diHETEs and might have severe pathologic consequences in children.

Effect of buflomedil on the neutrophil-endothelial cell interaction under inflammatory and hypoxia conditions.

In hypoxia/ischaemia and ischaemia/reperfusion, human neutrophils are likely to play an important role in the development of endothelial cell damage in the microcirculation. Buflomedil hypochloride improves the capillary perfusion in such related situations, evoking a possible effect upon neutrophils. Using in vitro models of cell adhesion, buflomedil decreased 100% of histamine related neutrophil adhesion (flow system) and partially inhibited adhesion after IL-1-4 hours (flow and stable systems). Hypoxia induced neutrophil adhesion (4 hours) was also reduced by buflomedil, which decreased the expression of P-selectin at the surface of endothelial cells. As adenosine (NECA) exhibited the same results in hypoxia and theophylline inhibited them, such results support an action of buflomedil presumably via the A2 receptor.

Plasma thrombomodulin: new approach of endothelium damage.

Endothelium damage is associated with thrombotic risk in a variety of diseases including atherosclerosis, gram negative sepsis, viral infections and neoplastic disease. Therefore, it appears necessary to find a mean for the clinical investigation for such a damage. Among the markers of these cells, thrombomodulin which is a membrane glycoprotein, seems to be of great interest for this purpose. Actually, thrombomodulin is also found in plasma, following an endothelial lesion. Plasma levels of thrombomodulin are increased in a certain number of pathologies associated with endothelium lesion: atheromatous arterial disease, disseminated intravascular coagulation syndrome and also in systemic lupus erythematosus where the levels of plasma thrombomodulin are related to the severity of the pathology. Moreover, previous in vitro studies confirm the fact that the release of thrombomodulin from the endothelial cell membrane occurs during the course of injury by activated leukocytes or hydrogen peroxide. So, one can suppose a prospective interest in the measurement of plasma thrombomodulin as a diagnostic tool for the approach of endothelium damage.

Thrombin-induced platelet aggregates have a dynamic structure. Time-dependent redistribution of glycoprotein IIb-IIIa complexes and secreted adhesive proteins.

The role of glycoprotein (GP) IIb-IIIa complexes and of adhesive proteins in mediating platelet aggregation is now well defined. However, less is known of the changes that occur once aggregation has begun. We report immunogold staining of thin sections of platelets or platelet aggregates, embedded in Lowicryl K4M, after the use of polyclonal antibodies to GP IIb or GP IIIa, fibrinogen (Fg), von Willebrand factor (vWF), and thrombospondin (TSP). Bound immunoglobulin G (IgG) was located by species-specific anti-IgG coupled to 5-nm gold particles and by electron microscopy. Initial experiments with platelet-rich plasma confirmed the feasibility of visualizing adhesive proteins between platelets in aggregates. Experiments then continued, using stirred suspensions of washed platelets incubated with alpha-thrombin. After 20 seconds, platelets were in contact without detectable release, although giant secretory vesicles containing adhesive proteins were seen. Internal pools of GP IIb-IIIa were progressively externalized within the aggregate. Secreted Fg was readily detected between platelets at 40 seconds. After 3 minutes, when most of the secretion had occurred, Fg had a patchwork-like distribution within the aggregate. After 6 minutes, zones with closely interspaced surface membranes, usually representing pseudopods, were dominant and Fg free. Results for vWF and TSP were similar to those for Fg. Nonetheless, GP IIb-IIIa complexes continued to be located between adjacent surface membranes throughout the aggregate. Thrombin-induced platelet aggregates were isolated, and sodium dodecyl sulfate-soluble extracts were obtained. Western blot experiments showed that, although fibrinopeptide A had been cleaved, degradation of adhesive proteins by platelet proteases had not occurred. These results emphasize that a platelet aggregate is a dynamic structure and suggest that not all surface-contact interactions are mediated by Fg or the other adhesive proteins tested in this study.