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Graphical systems models are powerful tools that can help facilitate hypothesis-driven ecotoxicogenomic research and aid in mechanistic interpretation of results. This paper describes a novel graphical model of the teleost brain-pituitary-gonadal (BPG) axis designed for ecotoxicogenomics research on endocrine-disrupting chemicals using small fish models. The model incorporates six compartments representing the major organs involved in the fish reproductive axis and depicts the interactions of over 105 proteins and 40 simple molecules, transcriptional regulation of 25 genes, and over 300 different reactions/ processes. Application of the model is illustrated in the context of a study examining effects of the competitive aromatase inhibitor, fadrozole, on gene expression in gonad, brain, and liver tissue of fathead minnows. Changes in mRNA transcript abundance were measured using a fathead minnow oligonucleotide microarray and quantitative real-time polymerase chain reaction. Gene expression changes observed in the ovaries of females exposed to 6.3 microg fadrozole/L for7 d were functionally consistent with fadrozole's mechanism of action, and expected compensatory responses of the BPG axis to fadrozole's effects. Furthermore, microarray results helped identify additional elements (genes/ proteins) that could be included in the model to potentially increase its predictive capacity. With proper recognition of

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Granule-bound starch synthase (GBSS), a product of the waxy gene in rice (Oryza sativa L.), is necessary for the synthesis of amylose in the endosperm. In an extended pedigree of 89 rice cultivars, we have previously shown that all cultivars with more than 18% amylose had the sequence AGGTATA at the leader intron 5' splice site, while all cultivars with a lower proportion of amylose had the sequence AGTTATA. This single-nucleotide polymorphism reduces the efficiency of GBSS pre-mRNA processing. It also results in alternate splicing at multiple sites, some of which have non-consensus sequences. Here we demonstrate that this same G-to-T polymorphism is also associated with differential sensitivity to temperature during the period of grain development. Cultivars with the sequence AGTTATA have a substantial increase in accumulation of mature GBSS transcripts at 18 degrees C compared to 25 or 32 degrees C. The selection of leader intron 5' splice sites is also affected by temperature in these cultivars. A 5' splice site -93 upstream from that used in high-amylose varieties predominates at 18 degrees C. At higher temperatures there is increased utilization of a 5' splice site at -I and a non-consensus site at +1. Potential implications of differential 5' splice site selection and associated differences in 3' splice site selection on transcript stability and translational efficiency are discussed.

Gene microarrays provide the field of ecotoxicology new tools to identify mechanisms of action of chemicals and chemical mixtures. Herein we describe the development and application of a 2,000-gene oligonucleotide microarray for the fathead minnow Pimephales promelas, a species commonly used in ecological risk assessments in North America. The microarrays were developed from various cDNA and subtraction libraries that we constructed. Consistency and reproducibility of the microarrays were documented by examining multiple technical replicates. To test application of the fathead minnow microarrays, gene expression profiles of fish exposed to 17beta-estradiol, a well-characterized estrogen receptor (ER) agonist, were examined. For these experiments, adult male fathead minnows were exposed for 24 h to waterborne 17beta-estradiol (40 or 100 ng/L) in a flow-through system, and gene expression in liver samples was characterized. Seventy-one genes were identified as differentially regulated by estradiol exposure. Examination of the gene ontology designations of these genes revealed patterns consistent with estradiol's expected mechanisms of action and also provided novel insights as to molecular effects of the estrogen. Our studies indicate the feasibility and utility of microarrays as a basis for understanding biological responses to chemical exposure in a model ecotoxicology test species.