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.

Detection of Simian Immunodeficiency Virus in Semen, Urethra, and Male Reproductive Organs during Efficient Highly Active Antiretroviral Therapy.

UNLABELLED: A number of men receiving prolonged suppressive highly active antiretroviral therapy (HAART) still shed human immunodeficiency virus (HIV) in semen. To investigate whether this seminal shedding may be due to poor drug penetration and/or viral production by long-lived cells within male genital tissues, we analyzed semen and reproductive tissues from macaques chronically infected with simian immunodeficiency virus mac251 (SIVmac251) who were treated for 4 months with HAART, which was intensified over the last 7 weeks with an integrase inhibitor. We showed that a subset of treated animals continued shedding SIV in semen despite efficient HAART. This shedding was not associated with low antiretroviral drug concentrations in semen or in testis, epididymis, seminal vesicles, and prostate. HAART had no significant impact on SIV RNA in the urethra, whereas it drastically reduced SIV RNA levels in the prostate and vas deferens and to a lesser extent in the epididymis and seminal vesicle. The only detectable SIV RNA-positive cells within the male genital tract after HAART were urethral macrophages. SIV DNA levels in genital tissues were not decreased by HAART, suggesting the presence throughout the male genital tract of nonproductively infected cells. In conclusion, our results demonstrate that 4 months of HAART induced variable and limited control of viral infection in the male reproductive organs, particularly in the urethra, and suggest that infected long-lived cells in the male genital tract may be involved in persistent seminal shedding during HAART. These results pave the way for further investigations of male genital organ infection in long-term-treated infected individuals. IMPORTANCE: A substantial subset of men receiving prolonged HAART suppressing viral loads in the blood still harbor HIV in semen, and cases of sexual transmission have been reported. To understand the origin of this persistence, we analyzed the semen and male reproductive tissues from SIV-infected macaques treated with HAART. We demonstrated that persistent seminal shedding was not linked to poor drug penetration in semen or semen-producing prostate, seminal vesicle, epididymis, and testis. We revealed that HAART decreased SIV RNA to various extents in all male genital organs, with the exception of the urethra, in which SIV RNA(+) macrophages were observed despite HAART. Importantly, HAART did not impact SIV DNA levels in the male genital organs. These results suggest that infection of male genital organs, and particularly the urethra, could be involved in the release of virus in semen during HAART.

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.

Development and validation of an LC/MS/MS assay for mycophenolic acid in human peripheral blood mononuclear cells.

The aim was to develop a LC/MS/MS method able to quantify mycophenolic acid (MPA) in the peripheral blood mononuclear cells (PBMCs) of transplanted patients. PBMCs were isolated from blood by a density gradient separation. The chromatographic separation was carried out on a Zorbax Stable Bond CN, 150 mmx2.1 mm, and MS/MS detection was performed after positive electrospray ionisation of the protonated parent ion. The calibration range was from 0.25 to 100 ng/sample. Extraction from the cells and ionisation recoveries reached 73.5 and 37.9%, respectively. Inaccuracy was always <10% with CVs<15%. MPA was stable at room temperature in the autosampler over 48 h and at -20 degrees C over 1.5 months. Application to clinical samples taken from patients treated with mycophenolate mofetil indicated that the method is suitable for measuring intracellular MPA.

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.

Direct determination of phosphorylated intracellular anabolites of stavudine (d4T) by liquid chromatography/tandem mass spectrometry.

The objective was to develop and validate a routine assay for active intracellular anabolites of stavudine (d4T), a nucleoside reverse transcriptase inhibitor in human PBMC, applicable to pharmacokinetic studies and treatment monitoring. This was achieved using liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS), which theoretically allies optimum sensitivity, specificity and high sample throughput. After cellular lysis in a Tris/methanol buffer, the extract spiked with 2[H(8)]-ATP (internal standard) is directly injected into the LC/MS/MS system. Phosphorylated metabolites of d4T as well as deoxythymidine-triphosphate, the competitor on the reverse transcriptase, are separated from d4T on a reverse-phase microbore column with ion pairing. The detection is performed in the multiple reaction monitoring (MRM) mode after drug ionisation in negative mode electrospray. The limit of quantitation for d4T-TP was 138 fmol per 7 mL blood (9.8 fmol per 10(6) cells) and CV% for repeatability and intermediate precision were lower than 15%. Stability of compounds was checked before and during the process of isolation of PBMC. Cellular samples from several d4T-treated patients were successfully analysed using this method and d4T-triphosphate and deoxythymidine triphosphate were recovered. In conclusion, we have developed and validated a routine LC/MS/MS method that allows the simultaneous determination of mono-, di- and triphosphorylated anabolites of d4T in PBMC as well as the natural corresponding triphosphate in one analysis. For the first time, the chain terminator ratio (d4T-TP/dT-TP) could be directly measured. This method can be used simply and routinely on more than 35 samples per day. Extension to other nucleoside analogues is under development.

Characterization of immunoreactive acetyl-Ser-Asp-Lys-Pro in human plasma and urine by liquid chromatography-electrospray mass spectrometry.

The tetrapeptide AcSDKP, a natural and specific substrate of angiotensin I-converting enzyme (ACE), is a negative regulator of hematopoiesis. AcSDKP has been measured in various biological media using an enzyme immunoassay (EIA), but its presence in human plasma and urine has not been formally established. By using immunoaffinity extraction and liquid chromatography-electrospray mass spectrometry, we demonstrate that AcSDKP-like immunoreactivity measured with EIA in plasma and urine samples from untreated, captopril- (an ACE inhibitor) and AcSDKP-treated subjects corresponds to AcSDKP. The present study confirms that AcSDKP is naturally present in human plasma and urine and that EIA is reliable for its measurement in such media.

Comparative methodologic study of NFkappaB activation in cultured endothelial cells.

The transcriptional regulatory protein nuclear factor kappaB (NFkappaB) participates in the control of gene expression of many modulators of the inflammatory and immune responses. Various activators trigger NFkappaB release and nuclear translocation after phosphorylation and proteolytic degradation of IkappaB. This study evaluated the abilities of fluorescence and confocal microscopies, laser scanning cytometry (LSC), electrophoretic mobility-shift assay (EMSA), and Western blotting to detect NFkappaB activation in endothelial cells (ECs) and to investigate the role of homocysteine (Hcy) in NFkappaB activation. ECs were treated with interleukin-1B (10 ng/mL) or Hcy thiolactone (1 and 5 mmol/L) as NFkappaB activators. Hcy, a thiol-containing amino acid, has been shown to directly damage ECs in vitro. Experimental evidence suggests that the atherogenic propensity associated with hyperhomocysteinemia results from EC dysfunction. When ECs were pretreated with an inhibitor (pyrrolidine dithiocarbamate, 100 micromol/L) or with staurosporine (5 microL/mL), no NFkappaB activation was observed. NFkappaB activation in ECs could be detected with all five techniques, clearly showing NFkappaB translocation from the cytoplasm to the nuclei. Confocal microscopy was more sensitive and less subjective than immunofluorescence microscopy. LSC was even more sensitive, specific, and reproducible. EMSA, the reference method, has the disadvantages of being radioactive, expensive, and time consuming. Western blot analysis detected the NFkappaB p50 subunit implicated in NFkappaB activation. The techniques usually used to detect NFkappaB activation in ECs are immunofluorescence microscopy and confocal microscopy, LSC, EMSA, and Western blot analysis, but none of them is ready for routine use.

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.

Fully automated determination of eserine N-oxide in human plasma using on-line solid-phase extraction with liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

A sensitive and entirely automated solid-phase extraction/liquid chromatography/electrospray ionization tandem mass spectrometric (SPE/LC/ESI-MS/MS) method was developed and validated for the determination of eserine N-oxide (ENO), a cholinesterase inhibitor-like physostigmine in human plasma, for use in pharmacokinetic studies. ENO is light-sensitive and the use of a fully on-line process increased the reliability of the assay. Plasma samples previously mixed with neostigmine bromide to prevent in vitro degradation, and tacrine as internal standard (IS), were directly injected into the SPE/LC/ESI-MS/MS system. MS software piloted the overall system. MS/MS detection of ENO and the IS was performed in the positive ion ESI mode using multiple reaction monitoring. The linear calibration curve for ENO ranged from 25 pg ml(-1) to 12.5 ng ml(-1). The limit of quantitation was 25 pg ml(-1) with 250 microl of plasma injected. Precision, accuracy and stability tests were within the acceptable range and just one analyst is required to analyze 50 unknown samples a day five days per week, from the preparation of the samples (i.e. thawing and centrifugation) to data processing. A pilot pharmacokinetic study in three healthy volunteers treated with 4.5 mg of ENO (Génésérine3((R))) showed that the method was suitable for pharmacokinetic studies in humans.

Schistosoma mansoni: in vitro adhesion of parasite eggs to the vascular endothelium. Subsequent inhibition by a monoclonal antibody directed to a carbohydrate epitope.

Schistosoma mansoni eggs come into direct contact with the vascular endothelium, particularly in the postcapillary venules of the mesenteric tract (oviposition site). We investigated the adhesion of eggs to endothelial cells in a static in vitro assay and in a flow-based in vitro assay. Live S. mansoni eggs rapidly attached, in a time-dependent manner, to the human endothelial cell line ECV 304, but not KOH-treated eggs. Activation of ECV monolayers with interleukin-1 promoted live S. mansoni eggs adhesion. An in vitro flow-based assay of human umbilical vein endothelial cells (HUVEC) showed the influence of wall shear stresses on the attachment of eggs to endothelial cells, particularly under postcapillary venule shear stress conditions. Interleukin-1 activation of HUVEC promoted adhesion between live eggs and endothelial cells. Higher wall shear stresses were needed to obtain the detachment of eggs from activated endothelial cells than control cells. Preincubation of interleukin-1-activated HUVEC, in a static in vitro assay, with monoclonal antibodies specific for intercellular adhesion molecule-1, E-selectin, and vascular cell adhesion molecule-1 significantly decreased adhesion of live eggs. Previous studies have shown that a monoclonal antibody specific for a schistosome carbohydrate epitope abundant in eggs is related to the Lewis X antigen. In this study, the anti-Lewis X-specific monoclonal antibody was used for adhesion-inhibition assays. Preincubation of eggs with this monoclonal antibody significantly decreased adhesion of live eggs to interleukin-1-activated HUVEC cultured in vitro. These results suggest that surface adhesion molecules, expressed by endothelial cells under conditions of interleukin-1 activation, directly participate in egg adhesion and that egg carbohydrate antigens play an important role in live S. mansoni egg adhesion to the vascular endothelium.

Development and in vivo assessment of a transdermal system for physostigmine.

A copolymer was developed as a transdermal (TD) system for physostigmine. The loading was carried out with a solution of physostigmine (PHY) base (20 mg/ml) in water/ethanol: 80/20 (v/v) at 40 degrees C for 3 h. The PHY load was 5.3 mg/cm2 (n = 3). Desorption carried out in vitro showed that 70% was desorbed during the first 6 h. More than 50% of the PHY was degraded within 45 min in skin homogenate. The TD was tested in vivo in rabbits during a 24 h experiment. PHY was quantified using a validated HPLC method. AUC0-24 h was 245.2 +/- 337.2 h.ng/ml. The mean pad flux reached 4.6 +/- 6.3 micrograms/cm2 from 0 to 24 h and, 24 h after the application of the pad, 110 micrograms/cm2 of PHY had been passed through the skin. After removed of the patch, plasma concentrations first increased from 15.8 +/- 28.6 ng/ml (at 24 h) to 21.4 +/- 36.7 ng/ml, then decreased with an elimination half-life of 0.7 +/- 0.2 h. AChE inhibition percentages increased from 6.5 +/- 2.3% to 16.0 +/- 27.7%. In vitro and in vivo studies in rabbit have shown that this system is suitable for further investigations in order to obtain a possible carrier for PHY therapy.

Magnesium isotopic abundance measurement in humans: comparison of two mass spectrometric methods.

Pharmacokinetic studies using stable isotopes of magnesium as tracers need to determine the isotopic abundance in biological media by means of mass spectrometry. Of mass spectrometric techniques, electronic impact-mass spectrometry (EI-MS) and inductively coupled plasma-mass spectrometry (ICP-MS) can be used. We have measured the isotopic abundance in plasma and urinary samples and compared the precision and accuracy of these two methods. Graphical representations showed that mean differences were close to 0, there was no obvious relationship between the difference and the mean, and no systematic bias was evidenced at low or high isotopic abundance. This was shown for isotopic abundance of either 25 Mg and 26 Mg. EI-MS is able to measure magnesium isotopic abundance with an intra-day precision between 0.14 and 0.45 per cent and with an inter-day precision between 0.20 and 1.23 per cent. ICP-MS exhibited an intra-day precision between 0.01 and 0.06 per cent and an inter-day precision between 0.01 and 0.15 per cent. Our results showed that, despite the similar isotopic abundances in biological samples obtained with the two methods, a large difference in precision clearly favours ICP-MS in studies of magnesium behaviour using stable isotopes.

Influence of hypoxia and hypoxia-reoxygenation on endothelial P-selectin expression.

P-selectin is an endothelial adhesion molecule involved in the initial step of the neutrophil recruitment. We investigated the effect of hypoxia (95% N2, 5% CO2) and of hypoxia-reoxygenation (95% air, 5% CO2) on the expression of P-selectin by human umbilical vein endothelial cells (HUVEC). P-selectin expression was detected by immunolabelling and quantified by flow cytometric analysis. Our data indicate that hypoxia induces an increase in P-selectin expression with a maximum reached after 90 minutes. A hypoxic exposure of 90 minutes results in a highly significant increase compared to normoxia (p < 0.001, n = 13). Furthermore, when a reoxygenation period follows 90 minutes of hypoxia, the initially elevated levels of P-selectin are dramatically enhanced with a maximum obtained after 60 minutes of reoxygenation.

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.