Over the last decade, the introduction of microarray technology has had a profound impact on gene expression research. The publication of studies with dissimilar or altogether contradictory results, obtained using different microarray platforms to analyze identical RNA samples, has raised concerns about the reliability of this technology. The MicroArray Quality Control (MAQC) project was initiated to address these concerns, as well as other performance and data analysis issues. Expression data on four titration pools from two distinct reference RNA samples were generated at multiple test sites using a variety of microarray-based and alternative technology platforms. Here we describe the experimental design and probe mapping efforts behind the MAQC project. We show intraplatform consistency across test sites as well as a high level of interplatform concordance in terms of genes identified as differentially expressed. This study provides a resource that represents an important first step toward establishing a framework for the use of microarrays in clinical and regulatory settings.

A number of perfluorinated alkyl acids including perfluorooctanoic acid (PFOA) elicit effects similar to peroxisome proliferator chemicals (PPC) in mouse and rat liver. There is strong evidence that PPC cause many of their effects linked to liver cancer through the nuclear receptor peroxisome proliferator-activated receptor alpha (PPAR alpha). To determine the role of PPAR alpha in mediating PFOA transcriptional events, we compared the transcript profiles of the livers of wild-type or PPAR alpha-null mice exposed to PFOA or the PPAR alpha agonist WY-14,643 (WY). After 7 days of exposure, 85% or 99.7% of the genes altered by PFOA or WY exposure, respectively were dependent on PPAR alpha. The PPAR alpha-independent genes regulated by PFOA included those involved in lipid homeostasis and xenobiotic metabolism. Many of the lipid homeostasis genes including acyl-CoA oxidase (Acox1) were also regulated by WY in a PPAR alpha-dependent manner. The increased expression of these genes in PPAR alpha-null mice may be partly due to increases in PPAR gamma expression upon PFOA exposure. Many of the identified xenobiotic metabolism genes are known to be under control of the nuclear receptor CAR (constitutive activated/androstane receptor) and the transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2). There was excellent correlation between the transcript profile of PPAR alpha-independent PFOA genes and those of activators of CAR including phenobarbital and 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) but not those regulated by the Nrf2 activator, dithiol-3-thione. These results indicate that PFOA alters most genes in wild-type mouse liver through PPAR alpha, but that a subset of genes are regulated by CAR and possibly PPAR gamma in the PPAR alpha-null mouse.

Perfluoroalkyl acids (PFAAs) are ubiquitous and persistent environmental contaminants. Compounds such as perfluoroocanoic acid (PFOA), perfluorooctane sulfonate (PFOS), perfluorononanoic acid (PFNA), and perfluorohexane sulfonate (PFHxS) are readily found in the tissues of humans and wildlife. While PFOA and PFOS have been the subject of numerous studies since they were first described over a decade ago, less is known about the biological activity of PFHxS and PFNA. Most PFAAs are activators of peroxisome proliferator-activated receptor α (PPARα), although the biological effects of these compounds are likely mediated by other factors in addition to PPARα. To evaluate the effects of PFHxS and PFNA, male wild-type and Pparα-null mice were dosed by oral gavage with PFHxS (3 or 10mg/kg/day), PFNA (1 or 3mg/kg/day), or vehicle for 7days, and liver gene expression was evaluated by full-genome microarrays. Gene expression patterns were then compared to historical in-house data for PFOA and PFOS in addition to the experimental hypolipidemic agent, WY-14,643. While WY-14,643 altered most genes in a PPARα-dependent manner, approximately 11-24% of regulated genes in PFAA-treated mice were independent of PPARα. The possibility that PFAAs regulate gene expression through other molecular pathways was evaluated. Using data available through a microarray database, PFAA gene expression profiles were found to exhibit significant similarity to profiles from mouse tissues exposed to agonists of the constitutive activated receptor (CAR), estrogen receptor α (ERα), and PPARγ. Human PPARγ and ERα were activated by all four PFAAs in trans-activation assays from the ToxCast screening program. Predictive gene expression biomarkers showed that PFAAs activate CAR in both genotypes and cause feminization of the liver transcriptome through suppression of signal transducer and activator of transcription 5B (STAT5B). These results indicate that, in addition to activating PPARα as a primary target, PFAAs also have the potential to activate CAR, PPARγ, and ERα as well as suppress STAT5B.

Perfluorooctanoic acid (PFOA) is a chemical used in the production of fluoropolymers. Its persistence in the environment and presence in humans and wildlife has raised health concerns. Liver tumor induction by PFOA is thought to be mediated in rodents by PPAR-α. A recent US EPA scientific advisory board questioned the contribution of PPAR-α in PFOA-induced liver tumors. Liver response in CD-1, SV/129 wild-type (WT), and PPAR-α knockout (KO) SV/129 mice was evaluated after seven daily treatments of PFOA-NH 4 + (1, 3, or 10 mg/kg, p.o.) or the prototype PPARα-agonist Wyeth 14,643 (WY, 50 mg/kg). Livers were examined by light and electron microscopy. Proliferation was quantified after PCNA immunostaining. PFOA treatment induced a dose-dependent increase in hepatocyte hypertrophy and labeling index (LI) similar to WY in WT mice. Ultrastructural alterations of peroxisome proliferation were similar between WY-treated and 10 mg/kg PFOA-treated WT mice. KO mice had a dose-dependent increase in hepatocyte vacuolation but increased LI only at 10 mg PFOA/kg. WY-treated KO mice were not different from KO control. These data suggest that PPAR-α is required for WY-and PFOA-induced cellular alterations in WT mouse liver. Hepatic enlargement observed in KO mice may be due to an accumulation of cytoplasmic vacuoles that contain PFOA.