The liver toxicity biomarker study phase I: markers for the effects of tolcapone or entacapone.

The Liver Toxicity Biomarker Study is a systems toxicology approach to discover biomarkers that are indicative of a drug's potential to cause human idiosyncratic drug-induced liver injury. In phase I, the molecular effects in rat liver and blood plasma induced by tolcapone (a "toxic" drug) were compared with the molecular effects in the same tissues by dosing with entacapone (a "clean" drug, similar to tolcapone in chemical structure and primary pharmacological mechanism). Two durations of drug exposure, 3 and 28 days, were employed. Comprehensive molecular analysis of rat liver and plasma samples yielded marker analytes for various drug-vehicle or drug-drug comparisons. An important finding was that the marker analytes associated with tolcapone only partially overlapped with marker analytes associated with entacapone, despite the fact that both drugs have similar chemical structures and the same primary pharmacological mechanism of action. This result indicates that the molecular analyses employed in the study are detecting substantial "off-target" markers for the two drugs. An additional interesting finding was the modest overlap of the marker data sets for 3-day exposure and 28-day exposure, indicating that the molecular changes in liver and plasma caused by short- and long-term drug treatments do not share common characteristics.

Minimal impact of excess iodate intake on thyroid hormones and selenium status in older New Zealanders.

OBJECTIVE: Iodine deficiency has re-emerged in New Zealand, while selenium status has improved. The aim of this study was to investigate the effects of excess iodine intake as iodate on thyroid and selenium status. METHODS: In a randomized controlled trial on older people (mean±s.d. 73±4.8 years; n=143), two groups received >50  mg iodine as iodate/day for 8 weeks because of supplement formulation error, either with 100  µg selenium (Se+highI) or without selenium (highI). Four other groups received 80  µg iodine as iodate/day with selenium (Se+lowI) or without selenium (lowI), selenium alone (Se+), or placebo. Thyroid hormones, selenium status, and median urinary iodine concentration (MUIC) were compared at weeks 0, 8, and 4 weeks post-supplementation. RESULTS: MUIC increased nine- and six-fold in Se+highI and highI groups, decreasing to baseline by week 12. Plasma selenium increased in selenium-supplemented groups (P<0.001). The level of increase in whole blood glutathione peroxidase (WBGPx) in the Se+highI group was smaller than Se+ (P=0.020) and Se+lowI (P=0.007) groups. The decrease in WBGPX in the highI group was greater than other non-selenium-supplemented groups, but differences were not significant. Ten of 43 participants exposed to excess iodate showed elevated TSH (hypothyroidism) at week 8. In all but two, TSH had returned to normal by week 12. In three participants, TSH decreased to <0.10  mIU/l (hyperthyroidism) at week 8, remaining low at week 12. CONCLUSIONS: Excess iodate induced hypothyroidism in some participants and hyperthyroidism in others. Most abnormalities disappeared after 4 weeks. Excess iodate reduced WBGPx activity and resulted in smaller increases in WBGPx after selenium supplementation.

Selenium and iodine supplementation: effect on thyroid function of older New Zealanders.

BACKGROUND: The New Zealand population has both marginal selenium status and mild iodine deficiency. Adequate intakes of iodine and selenium are required for optimal thyroid function. OBJECTIVE: The aim of the study was to determine whether low selenium and iodine status compromises thyroid function in an older New Zealand population. DESIGN: We investigated the effects of selenium and iodine supplementation in a double-blind, randomized, placebo-controlled trial in 100 Dunedin volunteers aged 60-80 y. Participants received 100 microg Se/d as l-selenomethionine, 80 microg I, 100 microg Se + 80 microg I, or placebo for 3 mo. Thyroid-stimulating hormone (TSH), free triiodothyronine (T(3)), free thyroxine (T(4)), thyroglobulin, plasma selenium, whole-blood glutathione peroxidase (GPx) activity, and urinary iodine concentrations (UICs) were measured. RESULTS: Plasma selenium (P < 0.0001) and whole-blood GPx activity (P<0.0001) increased from baseline to week 12 in the selenium and selenium plus iodine groups in comparison with the placebo group. Median UIC at baseline was 48 microg/L (interquartile range: 31-79 microg/L), which is indicative of moderate iodine deficiency. UIC increased in the iodine and selenium plus iodine groups and was significant only for the iodine group (P = 0.0014). Thyroglobulin concentration decreased by 24% and 13% of baseline in the iodine and selenium plus iodine groups in comparison with the placebo group (P = 0.009 and P = 0.108, respectively). No significant treatment effects were found for TSH, free T(3), free T(4), or ratio of T(3) to T(4). CONCLUSIONS: Additional selenium improved GPx activity but not the thyroid hormone status of older New Zealanders. Iodine supplementation alleviated the moderate iodine deficiency and reduced elevated thyroglobulin concentrations. No synergistic action of selenium and iodine was observed. The trial was registered at www.anzctr.org.au/registry/ as ACTRN012605000368639.

Methylation analysis by DNA immunoprecipitation (MeDIP).

Alteration in epigenetic regulation of gene expression is a common event in human cancer and developmental disease. CpG island hypermethylation and consequent gene silencing is observed for many genes involved in a diverse range of functions and pathways that become deregulated in the disease state. Comparative profiling of the methylome is therefore useful in disease gene discovery. The ability to identify epigenetic alterations on a global scale is imperative to understanding the patterns of gene silencing that parallel disease progression. Methylated DNA immunoprecipitation (MeDIP) is a technique that isolates methylated DNA fragments by immunoprecipitating with 5'-methylcytosine-specific antibodies. The enriched methylated DNA can then be analyzed in a locus-specific manner using PCR assay or in a genome-wide fashion by comparative genomic hybridization against a sample without MeDIP enrichment. This article describes the detailed protocol for MeDIP and hybridization of MeDIP DNA to a whole-genome tiling path BAC array.

The liver toxicity biomarker study: phase I design and preliminary results.

Drug-induced liver injury (DILI) is the primary adverse event that results in withdrawal of drugs from the market and a frequent reason for the failure of drug candidates in development. The Liver Toxicity Biomarker Study (LTBS) is an innovative approach to investigate DILI because it compares molecular events produced in vivo by compound pairs that (a) are similar in structure and mechanism of action, (b) are associated with few or no signs of liver toxicity in preclinical studies, and (c) show marked differences in hepatotoxic potential. The LTBS is a collaborative preclinical research effort in molecular systems toxicology between the National Center for Toxicological Research and BG Medicine, Inc., and is supported by seven pharmaceutical companies and three technology providers. In phase I of the LTBS, entacapone and tolcapone were studied in rats to provide results and information that will form the foundation for the design and implementation of phase II. Molecular analysis of the rat liver and plasma samples combined with statistical analyses of the resulting datasets yielded marker analytes, illustrating the value of the broad-spectrum, molecular systems analysis approach to studying pharmacological or toxicological effects.

Integrative genomic and gene expression analysis of chromosome 7 identified novel oncogene loci in non-small cell lung cancer.

Lung cancer accounts for over a quarter of cancer deaths, with non-small cell lung cancer (NSCLC) accounting for approximately 80% of cases. Several genome studies have been undertaken in both cell models of NSCLC and clinical samples to identify alterations underlying disease behaviour, and many have identified recurring aberrations of chromosome 7. The presence of recurring chromosome 7 alterations that do not span the well-studied oncogenes EGFR (at 7p11.2) and MET (at 7q31.2) has raised the hypothesis of additional genes on this chromosome that contribute to tumourigenesis. In this study, we demonstrated that multiple loci on chromosome 7 are indeed amplified in NSCLC, and through integrative analysis of gene dosage alterations and parallel gene expression changes, we identified new lung cancer oncogene candidates, including FTSJ2, NUDT1, TAF6, and POLR2J. Activation of these key genes was confirmed in panels of clinical lung tumour tissue as compared with matched normal lung tissue. The detection of gene activation in multiple cohorts of samples strongly supports the presence of key genes involved in lung cancer that are distinct from the EGFR and MET loci on chromosome 7.

Brazil nuts: an effective way to improve selenium status.

BACKGROUND: Brazil nuts provide a rich natural source of selenium, yet no studies have investigated the bioavailability of selenium in humans. OBJECTIVE: We investigated the efficacy of Brazil nuts in increasing selenium status in comparison with selenomethionine. DESIGN: A randomized controlled trial was conducted with 59 New Zealand adults. Participants consumed 2 Brazil nuts thought to provide approximately 100 mug Se, 100 mug Se as selenomethionine, or placebo daily for 12 wk. Actual intake from nuts averaged 53 mug Se/d (possible range: 20-84 mug Se). Plasma selenium and plasma and whole blood glutathione peroxidase (GPx) activities were measured at baseline and at 2, 4, 8, and 12 wk, and effects of treatments were compared. RESULTS: Plasma selenium increased by 64.2%, 61.0%, and 7.6%; plasma GPx by 8.3%, 3.4%, and -1.2%; and whole blood GPx by 13.2%, 5.3%, and 1.9% in the Brazil nut, selenomethionine, and placebo groups, respectively. Change over time at 12 wk in plasma selenium (P < 0.0001 for both groups) and plasma GPx activity in the Brazil nut (P < 0.001) and selenomethionine (P = 0.014) groups differed significantly from the placebo group but not from each other. The change in whole blood GPx activity was greater in the Brazil nut group than in the placebo (P = 0.002) and selenomethionine (P = 0.032) groups. CONCLUSION: Consumption of 2 Brazil nuts daily is as effective for increasing selenium status and enhancing GPx activity as 100 mug Se as selenomethionine. Inclusion of this high-selenium food in the diet could avoid the need for fortification or supplements to improve the selenium status of New Zealanders.

Fragmentation of leucine enkephalin as a function of laser fluence in a MALDI TOF-TOF.

The effects of laser fluence on ion formation in MALDI were studied using a tandem TOF mass spectrometer with a Nd-YAG laser and alpha-cyano hydrocinnamic acid matrix. Leucine enkephalin ionization and fragmentation were followed as a function of laser fluence ranging from the threshold of ion formation to the maximum available, that is, about 280-930 mJ/mm2. The most notable finding was the appearance of immonium ions at fluence values close to threshold, increasing rapidly and then tapering in intensity with the appearance of typical backbone fragment ions. The data suggest the presence of two distinct environments for ion formation. One is associated with molecular desorption at low values of laser fluence that leads to extensive immonium ion formation. The second becomes dominant at higher fluences, is associated initially with backbone type fragments, but, at the highest values of fluence, progresses to immonium fragments. This second environment is suggestive of ion desorption from large pieces of material ablated from the surface. Arrhenius rate law considerations were used to estimate temperatures associated with the onset of these two processes.

Assaying Bcr-Abl kinase activity and inhibition in whole cell extracts by phosphorylation of substrates immobilized on agarose beads.

There is a current and increasing demand for simple, robust, nonradioactive assays of protein tyrosine kinase activity with applications for clinical diagnosis and high-throughput screening of potential molecularly targeted therapeutic agents. One significant challenge is to detect and measure the activity of specific kinases with key roles in cell signaling as an approach to distinguish normal cells from cancer cells and as a means of evaluating targeted drug efficacy and resistance in cancer cells. Here, we describe a method in which kinase substrates fused to glutathione-S-transferase and immobilized on glutathione agarose beads are phosphorylated, eluted, and then assayed to detect kinase activity. The activity of recombinant, purified c-Abl kinase or Bcr-Abl kinase in whole cell extracts can be detected with equivalent specificity, sensitivity, and reproducibility. Similarly, inhibition of recombinant c-Abl or Bcr-Abl in cells or cell extracts by imatinib mesylate and other Bcr-Abl targeted kinase inhibitors is readily assayed. This simple kinase assay is sufficiently straightforward and robust for use in clinical laboratories and is potentially adaptable to high-throughput assay formats.

Tandem time-of-flight mass spectrometry.

A new tandem time-of-flight (TOF-TOF) instrument has been developed by modifying a standard matrix-assisted laser desorption ionization (MALDI)-TOF instrument to make high-performance, high-energy collision-induced dissociation (CID) MALDI tandem mass spectrometry (MS) a practical reality. To optimize fragment spectra quality, the selected precursor ion is decelerated before entering a floating collision cell and the potential difference between the source and the collision cell defines the collision energy of the ions. Standard operating conditions for tandem MS use a 1-kV collision energy with single-collision conditions and increased laser power for ion formation. Hence, both high- and low-energy fragments are observed in MALDI TOF-TOF spectra. On standard peptides, sensitivities down to 1 fmol are demonstrated. On a mixture of two solution tryptic digests at the 25-fmol level, 23 spectra were sufficient to result in proper database identification.

Intact protein analysis by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry.

Direct tandem mass spectrometric (MS/MS) analysis of small, singly charged protein ions by tandem time-of-flight mass spectrometry (TOFMS) is demonstrated for proteins up to a molecular mass of 12 kDa. The MALDI-generated singly charged precursor ions predominantly yield product ions resulting from metastable fragmentation at aspartyl and prolyl residues. Additional series of C-terminal sequence ions provide in some cases sufficient information for protein identification. The amount of sample required to obtain good quality spectra is in the high femtomolar to low picomolar range. Within this range, MALDI-MS/MS using TOF/TOF trade mark ion optics now provides the opportunity for direct protein identification and partial characterization without prior enzymatic hydrolysis.

De novo sequencing of peptides using MALDI/TOF-TOF.

The recently developed MALDI TOF-TOF instrument yields relatively complex but interpretable fragmentation spectra. When coupled with a straightforward sequence extension algorithm, it is possible to develop complete peptide sequences de novo from the spectra. This approach has been applied to a set of peptides derived from typtic digestion of electrophoretically separated sea urchin egg membrane proteins. When directed to proteins that have been described previously, the results were in essential agreement with those obtained by conventional data base searching approaches, with certain important exceptions. The present method detected errors in published sequences and was able to develop sequences from peptides differing in mass by one dalton (Da). These results show both the power of the present approach and the need for using de novo methods more frequently than may be otherwise appreciated.

Matrix-assisted laser desorption/ionization-tandem mass spectrometry with high resolution and sensitivity for identification and characterization of proteins.

Although peptide mass fingerprinting is currently the method of choice to identify proteins, the number of proteins available in databases is increasing constantly, and hence, the advantage of having sequence data on a selected peptide, in order to increase the effectiveness of database searching, is more crucial. Until recently, the ability to identify proteins based on the peptide sequence was essentially limited to the use of electrospray ionization tandem mass spectrometry (MS) methods. The recent development of new instruments with matrix-assisted laser desorption/ionization (MALDI) sources and true tandem mass spectrometry (MS/MS) capabilities creates the capacity to obtain high quality tandem mass spectra of peptides. In this work, using the new high resolution tandem time of flight MALDI-(TOF/TOF) mass spectrometer from Applied Biosystems, examples of successful identification and characterization of bovine heart proteins (SWISS-PROT entries: P02192, Q9XSC6, P13620) separated by two-dimensional electrophoresis and blotted onto polyvinylidene difluoride membrane are described. Tryptic protein digests were analyzed by MALDI-TOF to identify peptide masses afterward used for MS/MS. Subsequent high energy MALDI-TOF/TOF collision-induced dissociation spectra were recorded on selected ions. All data, both MS and MS/MS, were recorded on the same instrument. Tandem mass spectra were submitted to database searching using MS-Tag or were manually de novo sequenced. An interesting modification of a tryptophan residue, a "double oxidation", came to light during these analyses.

Off-line coupling of high-resolution capillary electrophoresis to MALDI-TOF and TOF/TOF MS.

High-resolution capillary electrophoresis has been coupled to MALDI-TOF and TOF/TOF MS through off-line vacuum deposition onto standard stainless steel MALDI targets. This off-line approach allowed the decoupling of the separation from the MS analysis, thus allowing each to be independently optimized in terms of time. Using BSA tryptic digest as a model sample, the deposited streaks, roughly 100-microm wide, were first analyzed in the MS mode, consuming only a fraction of the sample. After data analysis, segments of the deposited trace, containing unidentified peptides, as well as several species chosen for sequence confirmation, were reanalyzed in the MS/MS mode using MALDI-TOF/TOF MS. Additionally, it is shown that the shot-to-shot reproducibility of the vacuum-deposited trace (5% RSD) is 1 order of magnitude lower than that found for the standard dried droplet method. Moreover, a linear dependence of signal intensities (relative to an internal standard) over 3 orders of magnitude was found for a peptide sample with concentrations ranging from 1 to 1000 nM. This paper demonstrates the potential of off-line coupling of high-resolution separations to MALDI-MS and MALDI-MS/MS using vacuum deposition for the analysis of complex peptide mixtures from protein digests.