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Hematopoietic stem and progenitor cells (HSPC) can improve the long-term outcome of transplanted individuals and reduce the relapse rate. Valproic acid (VPA), an inhibitor of histone deacetylase, when combined with different cytokine cocktails, induces the expansion of CD34+ cell populations derived from cord blood (CB) and other sources. We evaluated the effect of VPA, in combination with thrombopoietin (TPO), on the viability and expansion of CB-HSPCs and on short- and long-term engraftability in the NOD/SCID mouse model. In vitro, VPA+TPO inhibited HSPC differentiation and preserved the CD34+ cell fraction; the self-renewal of the CD34+ TPO+VPA-treated cells was suggested by the increased replating efficiency. In vivo, short- and long-term engraftment was determined after 6 and 20 weeks. After 6 weeks, the median chimerism percentage was 13.0% in mice transplanted with TPO-treated cells and only 1.4% in those transplanted with TPO+VPA-treated cells. By contrast, after 20 weeks, the engraftment induced by the TPO+VPA-treated cells was three times more effective than that induced by TPO alone, and over ten times more effective compared to the short-term engraftment induced by the TPO+VPA-treated cells. The in vivo results are consistent with the higher secondary plating efficiency of the TPO+VPA-treated cells in vitro., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Reproductive toxicity, with its many targets and mechanisms, is a complex area of toxicology; thus, the screening and identification of reproductive toxicants is a main scientific challenge for the safety assessment of chemicals, including the European Regulation on Chemicals (REACH). Regulatory agencies recommend the implementation of the 3Rs principle (refinement, reduction, replacement) as well as of intelligent testing strategies, through the development of in vitro methods and the use of mechanistic information in the hazard identification and characterization steps of the risk assessment process. The EU Integrated Project ReProTect (6th Framework Programme) implemented an array of in vitro tests to study different building blocks of the mammalian reproductive cycle: methodological developments and results on male and female germ cells, prostate and placenta are presented.

Semicarbazide (SEM) is a by-product of the blowing agent azodicarbonamide, present in glass jar-sealed foodstuffs mainly baby foods. The pleiotropic in vivo SEM toxicological effects suggested to explore its possible role as endocrine modulator. Endocrine effects of SEM were assessed in vivo in male and female rats after oral administration for 28 days at 0, 40, 75, 140mg/kg bw pro die during the juvenile period. Vaginal opening and preputial separation were recorded. Concentration of sex steroid in blood, the ex vivo hepatic aromatase activity and testosterone catabolism were detected. The in vitro approach to test SEM role as (anti)estrogen or N-methyl-d-aspartate receptors (NMDARs)-(anti)agonist included different assays: yeast estrogenicity, MCF-7 proliferation, stimulation of the alkaline phosphatase activity in Ishikawa cells and LNCaP-based NMDAR interference assay. In vivo SEM-treated female rats showed delayed vaginal opening at all tested doses, whereas in males preputial separation was anticipated at SEM 40 and 75mg/kg and delayed at 140mg/kg, the latter effect probably due to the significantly decreased body weight gain seen at the higher dose in both sexes. Serum estrogen levels were dose-dependently reduced in treated females, whereas dehydrotestosterone serum levels were also decreased but a clear dose-response was not evidenced. Testosterone catabolism was altered in a gender-related way, aromatase activity was increased in treated males at 75 and 140mg/kg and in females in all dose groups. In the three estradiol-competitive assays, SEM showed a weak anti-estrogenic activity, whereas in the LNCaP-based NMDAR interference assay SEM activity resembled MK-801 antagonist effect. SEM appeared to act as an endocrine disrupter showing multiple and gender specific mechanisms of action(s). A possible cascade-mechanism of SEM on reproductive signalling pathways may be hypothesized. Such in vivo-in vitro approach appeared to be an useful tool to highlight SEM activity on endocrine homeostasis.

An isocratic RP-HPLC method for the determination of retinol and alpha-tocopherol in serum, with fluorescence and UV/VIS detection, respectively, was developed and validated according to international guidelines. Detection (retinol 0.015 mg/L, alpha-tocopherol 0.29 mg/L) and quantification (retinol 0.05 mg/L, alpha-tocopherol 0.95 mg/L) limits were determined. Repeatability was <3.3% and <2.9% and intermediate precision was <4.6% and <3.2%, for retinol and alpha-tocopherol, respectively. Certified reference materials were utilised to assess bias and guarantee traceability to SI units. Expanded uncertainties (retinol 8.9%; alpha-tocopherol 7.9%), estimated according to the EURACHEM/CITAC guide from method validation data, satisfied fit-for-purpose requirements based on biological variability.

Background: Immunoassays allow the specific detection and quantitation of biological molecules in complex samples at physiologically relevant concentrations. However, there are concerns over the comparability of such techniques when the same assay is performed by different operators or laboratories. An international intercomparison study was performed to assess the uncertainty involved in the estimation of a protein cytokine concentration using a fluorescent ELISA., Methods: The intercomparison study method was based on a non-competitive sandwich immunoassay with an enhancement step to generate a fluorescent readout. The intercomparison was performed in two phases, with the uncertainty of the instrument determined separately from that of the assay. The 11 laboratories participating in the study represented national metrology institutes or nominated expert laboratories., Results: Participants were asked to determine an undisclosed concentration of interferon using a supplied standard. The mean participant estimate and experimental standard deviation of the mean was 3.54+/-0.22 mg/L, with the spread of data ranging around +/-35% of the mean. The quantitation range of the ELISA and of participants' instruments displayed large variation that contributed to the overall uncertainty., Conclusions: Identified sources of uncertainty within the ELISA methodology included pipetting, data fitting, model selection and instrument/plate variation.

The opioid most commonly sold in the illicit market is heroin. This substance, classified as an analgesic narcotic drug, has an extremely short half-life, and it is rapidly metabolized to 6-monoacetyl-morphine and further to morphine. Morphine is principally metabolized by conjugation to morphine-3 and morphine-6 glucuronides. Morphine itself is a potent analgesic that is frequently used in the pharmacological intervention of cancer pain. The toxicological and clinical evaluation of heroin and morphine have stimulated pharmacokinetic studies in human and animal models. Although a number of methods exist to determine opiates and their metabolites, liquid chromatography (LC) appears to be the technique that can separate without any pretreatment the lipophilic and the hydrophilic analytes of the complete metabolic profile of heroin and/or morphine. Moreover, mass spectrometry (MS) used as a detector for liquid chromatography is unique, because it offers universality and selectivity. Furthermore, efforts have been made to develop LC/MS interfaces that could overcome the previous problem of poor sensitivity. For this reason, in recent years LC combined with MS has been applied to the analysis of opiates--parent drugs and metabolites--in biological fluids. This article reviews the existing literature on the determination, using liquid chromatography coupled to mass spectrometry, of opiate metabolites found in different biological matrices after the administration of the parent compounds.

Determination of the concentration of drugs and metabolites in biological fluids or matrices other than blood or urine (most commonly used in laboratory testing) may be of interest in certain areas of drug concentration monitoring. Saliva is the only fluid which can be used successfully as a substitute for blood in therapeutic drug monitoring, while an individual's past history of medication, compliance and drug abuse, can be obtained from drug analysis of the hair or nails. Drug concentrations in the bile and faeces can account for excretion of drugs and metabolites other than by the renal route. Furthermore, it is important that certain matrices (tears, nails, cerebrospinal fluid, bronchial secretions, peritoneal fluid and interstitial fluid) are analysed, as these may reveal the presence of a drug at the site of action; others (fetal blood, amniotic fluid and breast milk) are useful for determining fetal and perinatal exposure to drugs. Finally, drug monitoring in fluids such as cervical mucus and seminal fluid can be associated with morpho-physiological modifications and genotoxic effects. Drug concentration measurement in nonconventional matrices and fluids, although sometimes expensive and difficult to carry out, should therefore be considered for inclusion in studies of the pharmacokinetics and pharmacodynamics of new drugs.

The impact of stress and its neuroendocrine correlates on immune function are well established and individual variations could be attributed to modulation by personality characteristics. To assess the influence of everyday life stress and personality on neuroendocrine and immune function, we administered, to 18 healthy adults, the Minnesota Multiphasic Personality Inventory (MMPI) to assess their personality, the State-Trait Anxiety Inventory to measure anxiety, the Reaction Scheme Test to assess their coping reaction style, the Life Events Survey to assess the impact of stressful life events, and the Subjective Stress Questionnaire to assess perceived stress. The endocrine evaluation comprised prolactin, cortisol, and growth hormone plasma levels, while the immunological evaluation assessed T4, T8, and T11 lymphocyte percentages, as well as natural killer cell count and activity. All evaluations were made at baseline and after 8 months. We found a reduction of the T11 lymphocyte percentage to be accompanied by a reduction in the scores of the MMPI scale of Subtle Defensiveness and by an increase in the scores of the Social Introversion Scale. A positive correlation was found between prolactin and T4 lymphocyte percentage. These preliminary data show that some personality and endocrine measures correlate with immune function.

Sperm samples from 44 cigarette smokers and 50 nonsmokers attending an infertility clinic were examined by high-performance liquid chromatography (HPLC) assay and HPLC-mass spectrometry for the presence of nicotine (NIC), cotinine (COT), and trans-3'-hydroxycotinine (THOC) in seminal plasma. Smokers were found to have levels of COT and THOC in seminal plasma that were similar to those found in serum. The level of NIC was significantly increased in seminal plasma compared to serum. Total motility of spermatozoa was significantly and negatively correlated to COT and THOC levels in seminal plasma. Forward motility of spermatozoa was correlated only with cotinine semen levels. On the basis of these results, we suggest that the presence of tobacco smoke constituents in seminal plasma could provide a warning of the adverse effects of cigarette smoke on the physiology of reproduction.

A rapid and selective assay of nicotine, cotinine and trans-3'-hydroxycotinine in human serum, based on high-performance liquid chromatography with UV detection has been developed. The compounds were subjected to solid-phase extraction, using Extrelut 1 cartridges. Recoveries were ca. 95% for nicotine, 90% for cotinine and 50-55% for trans-3'-hydroxycotinine. The limit of quantitation observed with this method was 10 ng/ml for nicotine and 5 ng/ml for each of the metabolites. The compounds were also identified using high-performance liquid chromatography with particle beam mass spectrometry, to confirm their presence in human serum.

A liquid chromatographic-mass spectrometric method has been developed to determine busulfan concentrations in the cerebrospinal fluid and serum of some children undergoing bone marrow autotransplantation. After two liquid-liquid extraction steps with dichloromethane on a biological matrix, the separation of busulfan was carried out by isocratic reversed-phase chromatography. The mass spectrometric system was operated in electron-impact mode. Principal ions at m/z 175, 111 and 79 were observed for busulfan, but only m/z 175 was chosen for the quantification of the analyte. The retention time of busulfan was 2.5 min. The detection limit of 100 ng/ml allowed the determination of cerebrospinal fluid and serum busulfan concentrations during the four days of high-dose (1 mg/kg) treatment prior to autotransplantation in five child patients.

The relationship between nicotine intake and steady-state cotinine concentration was studied in a sample of 125 subjects who smoked their usual brands of cigarettes. Nicotine and tar yield of cigarettes was determined with a smoking machine, under standardized conditions. Blood was drawn about 8 hours after the last cigarette was smoked and serum cotinine was measured by high performance liquid chromatography. Cotinine levels ranged from 11 to 400 ng/ml, and nicotine daily intake ranged from 1 to 33 mg/day. Regression analysis and the correlation coefficient, r = 0.919, significant at p less than 0.0001, showed that steady-state cotinine level was linearly and directly related to daily available nicotine, with an increase in correlation coefficient directly related to the increase in tar and nicotine yield. From the findings we also conclude that smokers of low-tar cigarettes do not tend to compensate for lower yields of nicotine.

A simple and reliable reversed-phase high-performance liquid chromatographic method with coulometric detection is described for the quantitation of naltrexone and its metabolite, 6 beta-naltrexol, in plasma samples of healthy volunteers who received orally 50 mg of naltrexone. The analytes and the internal standard, naloxone, are extracted with an octadecyl solid-phase extraction column before chromatography. The mobile phase is 0.01 M potassium phosphate (pH 3)-acetonitrile (85:15, v/v) and it is pumped at 0.8 ml/min. The coulometric detector is formed by two electrodes set at +0.20 V and +0.70 V, with a palladium reference electrode. The limit of quantitation observed was 5 ng/ml for both naltrexone and 6 beta-naltrexol. This method can be used to investigate pharmacokinetic parameters of different pharmaceutical preparations of this opioid antagonist.