Assessment of the respiratory sensitization potential of proteins using an enhanced mouse intranasal test (MINT).

There remains a need for a simple and predictive animal model to identify potential respiratory sensitizers. The mouse intranasal test (MINT) was developed to assess the relative allergic potential of detergent enzymes, however, the experimental endpoints were limited to evaluation of antibody levels. The present study was designed to evaluate additional endpoints (serum and allergic antibody levels, pulmonary inflammation and airway hyperresponsiveness (AHR)) to determine their value in improving the predictive accuracy of the MINT. BDF1 mice were intranasally instilled on days 1, 3, 10, 17 and 24 with subtilisin, ovalbumin, betalactoglobulin, mouse serum albumin or keyhole limpet hemocyanin; challenged with aerosolized methacholine or the sensitizing protein on day 29 to assess AHR, and sacrificed on day 29 or 30. Under the conditions of this study, evaluation of AHR did not improve the predictive power of this experimental model. Allergic antibody responses and IgG isotype characterization proved to be the most sensitive and reliable indicators of the protein allergenic potential with BAL responses providing additional insight. These data highlight that the evaluation of the respiratory sensitization potential of proteins can be best informed when multiple parameters are evaluated and that further improvements and refinements of the assay are necessary.

Protocols for the assurance of microarray data quality and process control.

Microarrays represent a powerful technology that provides the ability to simultaneously measure the expression of thousands of genes. However, it is a multi-step process with numerous potential sources of variation that can compromise data analysis and interpretation if left uncontrolled, necessitating the development of quality control protocols to ensure assay consistency and high-quality data. In response to emerging standards, such as the minimum information about a microarray experiment standard, tools are required to ascertain the quality and reproducibility of results within and across studies. To this end, an intralaboratory quality control protocol for two color, spotted microarrays was developed using cDNA microarrays from in vivo and in vitro dose-response and time-course studies. The protocol combines: (i) diagnostic plots monitoring the degree of feature saturation, global feature and background intensities, and feature misalignments with (ii) plots monitoring the intensity distributions within arrays with (iii) a support vector machine (SVM) model. The protocol is applicable to any laboratory with sufficient datasets to establish historical high- and low-quality data.

Normalization of two-channel microarray experiments: a semiparametric approach.

MOTIVATION: An important underlying assumption of any experiment is that the experimental subjects are similar across levels of the treatment variable, so that changes in the response variable can be attributed to exposure to the treatment under study. This assumption is often not valid in the analysis of a microarray experiment due to systematic biases in the measured expression levels related to experimental factors such as spot location (often referred to as a print-tip effect), arrays, dyes, and various interactions of these effects. Thus, normalization is a critical initial step in the analysis of a microarray experiment, where the objective is to balance the individual signal intensity levels across the experimental factors, while maintaining the effect due to the treatment under investigation. RESULTS: Various normalization strategies have been developed including log-median centering, analysis of variance modeling, and local regression smoothing methods for removing linear and/or intensity-dependent systematic effects in two-channel microarray experiments. We describe a method that incorporates many of these into a single strategy, referred to as two-channel fastlo, and is derived from a normalization procedure that was developed for single-channel arrays. The proposed normalization procedure is applied to a two-channel dose-response experiment.

Comparative analysis of dioxin response elements in human, mouse and rat genomic sequences.

Comparative approaches were used to identify human, mouse and rat dioxin response elements (DREs) in genomic sequences unambiguously assigned to a nucleotide RefSeq accession number. A total of 13 bona fide DREs, all including the substitution intolerant core sequence (GCGTG) and adjacent variable sequences, were used to establish a position weight matrix and a matrix similarity (MS) score threshold to rank identified DREs. DREs with MS scores above the threshold were disproportionately distributed in close proximity to the transcription start site in all three species. Gene expression assays in hepatic mouse tissue confirmed the responsiveness of 192 genes possessing a putative DRE. Previously identified functional DREs in well-characterized AhR-regulated genes including Cyp1a1 and Cyp1b1 were corroborated. Putative DREs were identified in 48 out of 2437 human-mouse-rat orthologous genes between -1500 and the transcriptional start site, of which 19 of these genes possessed positionally conserved DREs as determined by multiple sequence alignment. Seven of these nineteen genes exhibited 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated regulation, although there were significant discrepancies between in vivo and in vitro results. Interestingly, of the mouse-rat orthologous genes with a DRE between -1500 and +1500, only 37% had an equivalent human ortholog. These results suggest that AhR-mediated gene expression may not be well conserved across species, which could have significant implications in human risk assessment.

Temporal- and dose-dependent hepatic gene expression changes in immature ovariectomized mice following exposure to ethynyl estradiol.

Temporal- and dose-dependent changes in hepatic gene expression were examined in immature ovariectomized C57BL/6 mice gavaged with ethynyl estradiol (EE), an orally active estrogen. For temporal analysis, mice were gavaged every 24 h for 3 days with 100 microg/kg EE or vehicle and liver samples were collected at 2, 4, 8, 12, 24 and 72 h. Gene expression was monitored using custom cDNA microarrays containing 3067 genes/ESTs of which 393 exhibited a change at one or more time points. Functional gene annotation extracted from public databases associated temporal gene expression changes with growth and proliferation, cytoskeletal and extracellular matrix responses, microtubule-based processes, oxidative metabolism and stress, and lipid metabolism and transport. In the dose-response study, hepatic samples were collected 24 h following treatment with 0, 0.1, 1, 10, 100 or 250 microg/kg EE. Thirty-nine of the 79 genes identified as differentially regulated at 24 h in the time course study exhibited a dose-response relationship with an average ED50 value of 47 +/- 3.5 microg/kg. Comparative analysis indicated that many of the identified temporal and dose-dependent hepatic responses are similar to EE-induced uterine responses reported in the literature and in a companion study using the same animals. Results from these studies confirm that the liver is a highly estrogen responsive tissue that exhibits a number of common responses shared with the uterus as well as distinct estrogen-mediated profiles. These data will further aid in the elucidation of the mechanisms of action of estrogens in the liver as well as in other classical and non-classical estrogen responsive tissues.

Recombinant human P450 forms 1A1, 1A2, and 1B1 catalyze the bioactivation of heterocyclic amine mutagens in Escherichia coli lacZ strains.

Three recombinant human P450 enzymes, forms 1A1, 1A2, and 1B1, were coexpressed with NADPH-cytochrome P450 reductase in an E. coli lacZ strain suitable for detection of the mutagenicity of heterocyclic and aromatic amines. The resulting strains expressed the recombinant P450 holoenzymes at high levels. MeIQ (2-amino-3,4-dimethylimidazo[4,5-f]quinoline) was activated effectively by P450 1A2, weakly by P450 1A1, and not detectably by P450 1B1. MeIQx (2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline) and Trp-P-2 (3-amino-1-methyl-5H-pyrido[4,3-b]indole) were activated by all three enzymes, with form 1A2 the most effective. These strains facilitate analysis of the substrate specificity of human P450 forms that participate in the metabolic activation of carcinogens.