Comparing next-generation sequencing and microarray technologies in a toxicological study of the effects of aristolochic acid on rat kidneys.

RNA-Seq has been increasingly used for the quantification and characterization of transcriptomes. The ongoing development of the technology promises the more accurate measurement of gene expression. However, its benefits over widely accepted microarray technologies have not been adequately assessed, especially in toxicogenomics studies. The goal of this study is to enhance the scientific community's understanding of the advantages and challenges of RNA-Seq in the quantification of gene expression by comparing analysis results from RNA-Seq and microarray data on a toxicogenomics study. A typical toxicogenomics study design was used to compare the performance of an RNA-Seq approach (Illumina Genome Analyzer II) to a microarray-based approach (Affymetrix Rat Genome 230 2.0 arrays) for detecting differentially expressed genes (DEGs) in the kidneys of rats treated with aristolochic acid (AA), a carcinogenic and nephrotoxic chemical most notably used for weight loss. We studied the comparability of the RNA-Seq and microarray data in terms of absolute gene expression, gene expression patterns, differentially expressed genes, and biological interpretation. We found that RNA-Seq was more sensitive in detecting genes with low expression levels, while similar gene expression patterns were observed for both platforms. Moreover, although the overlap of the DEGs was only 40-50%, the biological interpretation was largely consistent between the RNA-Seq and microarray data. RNA-Seq maintained a consistent biological interpretation with time-tested microarray platforms while generating more sensitive results. However, there is clearly a need for future investigations to better understand the advantages and limitations of RNA-Seq in toxicogenomics studies and environmental health research.

The balance of reproducibility, sensitivity, and specificity of lists of differentially expressed genes in microarray studies.

BACKGROUND: Reproducibility is a fundamental requirement in scientific experiments. Some recent publications have claimed that microarrays are unreliable because lists of differentially expressed genes (DEGs) are not reproducible in similar experiments. Meanwhile, new statistical methods for identifying DEGs continue to appear in the scientific literature. The resultant variety of existing and emerging methods exacerbates confusion and continuing debate in the microarray community on the appropriate choice of methods for identifying reliable DEG lists. RESULTS: Using the data sets generated by the MicroArray Quality Control (MAQC) project, we investigated the impact on the reproducibility of DEG lists of a few widely used gene selection procedures. We present comprehensive results from inter-site comparisons using the same microarray platform, cross-platform comparisons using multiple microarray platforms, and comparisons between microarray results and those from TaqMan - the widely regarded "standard" gene expression platform. Our results demonstrate that (1) previously reported discordance between DEG lists could simply result from ranking and selecting DEGs solely by statistical significance (P) derived from widely used simple t-tests; (2) when fold change (FC) is used as the ranking criterion with a non-stringent P-value cutoff filtering, the DEG lists become much more reproducible, especially when fewer genes are selected as differentially expressed, as is the case in most microarray studies; and (3) the instability of short DEG lists solely based on P-value ranking is an expected mathematical consequence of the high variability of the t-values; the more stringent the P-value threshold, the less reproducible the DEG list is. These observations are also consistent with results from extensive simulation calculations. CONCLUSION: We recommend the use of FC-ranking plus a non-stringent P cutoff as a straightforward and baseline practice in order to generate more reproducible DEG lists. Specifically, the P-value cutoff should not be stringent (too small) and FC should be as large as possible. Our results provide practical guidance to choose the appropriate FC and P-value cutoffs when selecting a given number of DEGs. The FC criterion enhances reproducibility, whereas the P criterion balances sensitivity and specificity.

Reproducible and reliable microarray results through quality control: good laboratory proficiency and appropriate data analysis practices are essential.

Over a few short years, microarray gene expression profiling has permeated most areas of biomedical research. Microarrays are now poised to enter the more demanding realm of clinical applications. The prospect of using microarray data to derive biomarkers of disease or toxicity, predict prognosis, or select treatments raises the validity and reliability bar substantially higher. The potential future payoffs are huge in terms of faster approval of more efficacious and safer medical interventions, and a more personalized implementation of them. Arriving at the future sooner rather than later is the motivation for the FDA-led MicroArray Quality Control (MAQC) project. The widespread collaboration aims to assess achievable technical performance of microarrays and capabilities and limitations of methods for microarray data analysis.

Microarray scanner calibration curves: characteristics and implications.

BACKGROUND: Microarray-based measurement of mRNA abundance assumes a linear relationship between the fluorescence intensity and the dye concentration. In reality, however, the calibration curve can be nonlinear. RESULTS: By scanning a microarray scanner calibration slide containing known concentrations of fluorescent dyes under 18 PMT gains, we were able to evaluate the differences in calibration characteristics of Cy5 and Cy3. First, the calibration curve for the same dye under the same PMT gain is nonlinear at both the high and low intensity ends. Second, the degree of nonlinearity of the calibration curve depends on the PMT gain. Third, the two PMTs (for Cy5 and Cy3) behave differently even under the same gain. Fourth, the background intensity for the Cy3 channel is higher than that for the Cy5 channel. The impact of such characteristics on the accuracy and reproducibility of measured mRNA abundance and the calculated ratios was demonstrated. Combined with simulation results, we provided explanations to the existence of ratio underestimation, intensity-dependence of ratio bias, and anti-correlation of ratios in dye-swap replicates. We further demonstrated that although Lowess normalization effectively eliminates the intensity-dependence of ratio bias, the systematic deviation from true ratios largely remained. A method of calculating ratios based on concentrations estimated from the calibration curves was proposed for correcting ratio bias. CONCLUSION: It is preferable to scan microarray slides at fixed, optimal gain settings under which the linearity between concentration and intensity is maximized. Although normalization methods improve reproducibility of microarray measurements, they appear less effective in improving accuracy.

Cross-platform comparability of microarray technology: intra-platform consistency and appropriate data analysis procedures are essential.

BACKGROUND: The acceptance of microarray technology in regulatory decision-making is being challenged by the existence of various platforms and data analysis methods. A recent report (E. Marshall, Science, 306, 630-631, 2004), by extensively citing the study of Tan et al. (Nucleic Acids Res., 31, 5676-5684, 2003), portrays a disturbingly negative picture of the cross-platform comparability, and, hence, the reliability of microarray technology. RESULTS: We reanalyzed Tan's dataset and found that the intra-platform consistency was low, indicating a problem in experimental procedures from which the dataset was generated. Furthermore, by using three gene selection methods (i.e., p-value ranking, fold-change ranking, and Significance Analysis of Microarrays (SAM)) on the same dataset we found that p-value ranking (the method emphasized by Tan et al.) results in much lower cross-platform concordance compared to fold-change ranking or SAM. Therefore, the low cross-platform concordance reported in Tan's study appears to be mainly due to a combination of low intra-platform consistency and a poor choice of data analysis procedures, instead of inherent technical differences among different platforms, as suggested by Tan et al. and Marshall. CONCLUSION: Our results illustrate the importance of establishing calibrated RNA samples and reference datasets to objectively assess the performance of different microarray platforms and the proficiency of individual laboratories as well as the merits of various data analysis procedures. Thus, we are progressively coordinating the MAQC project, a community-wide effort for microarray quality control.

13-week dietary toxicity study of ammonium perfluorooctanoate (APFO) in male rats.

Ammonium perfluorooctanoate is a perfluorinated carboxylate that is used commercially as a processing aid in the production of fluorinated polymers. Perfluorooctanoate (PFOA) has been found in human blood of the general population from exogenous sources. This report presents the results of a 13-week dietary toxicity study in male rats and was designed to identify potential target organ(s), dose response, and to explore possible relationships of PPARalpha activation to potential liver effects and hormonal changes. Rats were fed dietary levels of 0, 1, 10, 30, and 100 ppm (equivalent to 0, 0.06, 0.64, 1.94, and 6.5 mg/kg/day) for 13 weeks. A control group pair-fed adjusted to the 100 ppm level and groups allowed to recover for 8 weeks were included. Sacrifices were conducted after 4, 7, and 13 weeks of feeding and after 8 weeks of recovery. At each sacrifice, gross and histopathology was conducted on selected tissues and measurements of hepatic palmitoyl CoA oxidase (PCoAO), as well as serum estradiol, luteinizing hormone, testosterone, and PFOA were determined. There were no clinical signs or mortality. Body weight gains were reduced in the 100 ppm dose group. Liver weights (absolute and relative), PCoAO activity, and hepatocyte hypertrophy (minimal to mild) were increased in the 10 ppm dose group and above and were reversible in recovery. Under the study conditions, hormone levels appeared unchanged. PFOA serum concentrations increased in a dose-related fashion, appeared to reach steady-state by test week 5, and declined rapidly through the recovery period. Serum PFOA concentrations at the end of the treatment period were 7.1, 41, 70, and 138 microg/mL in the 1, 10, 30 and 100 ppm dose groups. The study no effect level was 1 ppm (0.06 microg/mg) with doses of 10 ppm (0.64 microg/mg) and higher producing adaptive and reversible liver changes.

The toxicology of perfluorooctanoate.

PFOA is a peroxisome proliferator (PPAR agonist) and exerts morphological and biochemical effects characteristic of PPAR agonists. These effects include increased beta-oxidation of fatty acids, increases in several cytochrome P-450 (CYP450)-mediated reactions, and inhibition of the secretion of very low-density lipoproteins and cholesterol from the liver. These effects on lipid metabolism and transport result in a reduction of cholesterol and triglycerides in serum and an accumulation of lipids in the liver. The triad of tumors observed (liver, Leydig cell, and pancreatic acinar-cell) is typical of many PPAR agonists and is believed to involve nongenotoxic mechanisms. The hepatocellular tumors observed in rats are likely to have been the result of the activation of the peroxisome proliferator activated receptor alpha (PPARalpha). The tumors observed in the testis (Leydig-cell) have been hypothesized to be associated with an increased level of serum estradiol in concert with testicular growth factors. The mechanism responsible for the acinar-cell tumors of the pancreas in rats remains the subject of active investigation. The mechanism resulting in the hepatocellular tumors in rats (PPARalpha activation) is not likely to be relevant to humans. Similarly, the proposed mechanism for Leydig-cell tumor formation is of questionable relevance to humans. Acinar tumors of the pancreas are rare in humans, and the relevance of the these tumors, as found in rats, to humans is uncertain. Epidemiological investigations and medical surveillance of occupationally exposed workers have not found consistent associations between PFOA exposure and adverse health effects.

Phytoestrogens and mycoestrogens bind to the rat uterine estrogen receptor.

Consumption of phytoestrogens and mycoestrogens in food products or as dietary supplements is of interest because of both the potential beneficial and adverse effects of these compounds in estrogen-responsive target tissues. Although the hazards of exposure to potent estrogens such as diethylstilbestrol in developing male and female reproductive tracts are well characterized, less is known about the effects of weaker estrogens including phytoestrogens. With some exceptions, ligand binding to the estrogen receptor (ER) predicts uterotrophic activity. Using a well-established and rigorously validated ER-ligand binding assay, we assessed the relative binding affinity (RBA) for 46 chemicals from several chemical structure classes of potential phytoestrogens and mycoestrogens. Although none of the test compounds bound to ER with the affinity of the standard, 17beta-estradiol (E(2)), ER binding was found among all classes of chemical structures (flavones, isoflavones, flavanones, coumarins, chalcones and mycoestrogens). Estrogen receptor relative binding affinities were distributed across a wide range (from approximately 43 to 0.00008; E(2) = 100). These data can be utilized before animal testing to rank order estimates of the potential for in vivo estrogenic activity of a wide range of untested plant chemicals (as well as other chemicals) based on ER binding.