High-density arrays of DNA bound to solid substrates offer a powerful approach to identifying changes in gene expression in response to toxicants. While DNA arrays have been used to explore qualitative changes in gene regulation, less attention has focused on the quantitative aspects of this technology. Arrays containing expressed sequence tags for xenobiotic metabolizing enzymes, proteins associated with glutathione regulation, DNA repair enzymes, heat shock proteins, and housekeeping genes were used to examine gene expression in response to ␤-naphthoflavone (␤-NF). Upregulation of cytochrome P4501a1 (Cyp1a1) and 1a2 in mouse liver was maximal 8 h after ␤-NF administration. Significant upregulation of Cyp1a2 was noted at ␤-NF doses as low as 0.62 and 1.2 mg/kg when gene expression was measured by microarray or Northern blotting, respectively. Maximal Cyp1a2 induction is 5-fold by Northern analysis and 10-fold by microarray. Induction of Cyp1a1 was 15-and 20-fold by Northern and microarray analysis, respectively. The coefficient of variation for spot to spot and slide to slide comparisons was <15%; this variability was smaller than interanimal variability (18 -60%). Comparison of mRNA expression in control animals indicated that there are differences in labeling/detection associated with Cy3/Cy5 dyes; accordingly, experiments must include methods for establishing baseline signals for all genes. We conclude that the dynamic range and sensitivity of DNA microarrays on glass slides is comparable to Northern blotting analysis and that variability of the data introduced during spotting and hybridization is less than the interanimal variability.

Toxicity testing of unknown chemicals currently uses a number of short-term bioassays. These tests are costly and time consuming, require large numbers of animals, and generally focus on a single end point. The recent development of DNA arrays provides a potential mechanism for increasing the efficiency of standard toxicity testing through genome-wide assessments of gene regulation. In this study, we used DNA arrays containing 148 genes for xenobiotic metabolizing enzymes, DNA repair enzymes, heat shock proteins, cytokines, and housekeeping genes to examine gene expression patterns in the liver in response to cadmium chloride, benzo(a)pyrene (BaP), and trichloroethylene (TCE). Dose-response studies were carried out in mice for each chemical; each produced a unique pattern of gene induction. As expected, CdCl2 markedly up-regulated metallothionine I and II (5- to 10,000-fold at the highest doses) and several of the heat shock/stress response proteins and early response genes. In contrast, administration of BaP up-regulated only Cyp1a1 and Cyp1a2 genes and produced no significant increases in any of the stress response genes or any of the DNA repair genes present on the array. Likewise, TCE-induced gene induction was highly selective; only Hsp 25 and 86 and Cyp2a were up-regulated at the highest dose tested. Microarray analysis with a highly focused set of genes is capable of discriminating between different classes of toxicants and has potential for differentiating highly noxious versus more subtle toxic agents. These data suggest that use of microarrays to evaluate the potential hazards of unknown chemicals or chemical mixtures must include multiple doses and time points to provide effective assessments of potential toxicity of these substances.

No abstract

2-DE is a powerful separation method for complex protein mixtures. However, large intergel variations in spot intensity limit its use for quantitative proteomics studies. To address this issue, we developed a fluorescent internal protein standard for use in 2-DE analysis. Protein samples are spiked with an Alexa-labeled internal standard (ALIS) prior to separation with 2-DE. Due to the high extinction coefficient of the Alexa-fluor, incorporation of 0.1% of total protein is sufficient to allow visualization of the internal standard yet low enough to avoid interference in subsequent quantification and identification steps. Following 2-DE, total proteins are visualized with fluorescent postelectrophoretic stains spectrally separated from ALIS. Four protein stains, Deep Purple, Sulforhodamine G, ruthenium II-tris(bathophenanthroline disulfonate) (RuTBS), and SYPRO Ruby, including improved purification and staining protocols for RuTBS and ten-fold dilutions of SYPRO Ruby were evaluated. All staining protocols were compatible with the ALIS method and had similar LODs (1-4 ng) and dynamic ranges (10(3)). ALIS is a powerful normalization method for quantitative 2-DE which avoids potential problems associated with dual spot migration patterns observed in the DIGE method. Furthermore, ALIS provides significantly improved normality in the distribution of spot abundance-variance compared to normalization through division by the total spot volume.

No abstract