The aryl hydrocarbon receptor (Ahr), a member of the large basic helix-loop-helix (bHLH) and PAS homology domain superfamily, is a highly conserved transcriptional regulator involved in mammalian development. In the present study, a murine metanephros organ culture system was employed to evaluate the role of the Ahr signaling in nephrogenesis in vitro. Ahr and Wilms' tumor suppressor (wt1) mRNAs were detected by in situ hybridization and RT-PCR during the course of renal development. Treatment with 3 lm BaP, a hydrocarbon ligand of Ahr, inhibited glomerulogenesis and branching morphogenesis of metanephric kidneys. Deficits in the epithelialization of mesenchymal cells were evidenced by inhibition of the formation of podocyte foot processes and glomerular basement membranes. Hydrocarbon treatment markedly induced ÀKTS wt1 splice variants, although total wt1 mRNA levels remained unchanged. A significant decrease in total WT1 protein was observed by both immunocytochemistry and Western analysis in cultures challenged with BaP compared to controls. Comparison of metanephric cultures from Ahr+/+ and AhrÀ/À mice showed that Ahr is involved in kidney development, and required for BaP-induced deficits in nephrogenesis. These results indicate that ligand activation of Ahr signaling disrupts nephrogenesis in vitro, and that this response involves modulation of wt1 alternative splicing and post-transcriptional control.

The co-expression of genes coupled to additive probabilistic relationships was used to identify gene sets predictive of the complex biological interactions regulated by ligands of the aryl hydrocarbon receptor ((Italic)Ahr(/Italic)). To maximize the number of possible gene-gene combinations, data sets from murine embryonic kidney, fetal heart, and vascular smooth muscle cells challenged (Italic)in vitro(/Italic) with ligands of the (Italic)Ahr(/Italic) were used to create predictor/training data sets. Biologically relevant gene predictor sets were calculated for (Italic)Ahr(/Italic), cytochrome P450 1B1, insulin-like growth factor-binding protein-5, lysyl oxidase, and osteopontin. Transcript levels were categorized into ternary expressions and target genes selected from the data set and tested for all possible combinations using three gene sets as predictors of transitional level. The goodness of prediction for each set was quantified using a multivariate nonlinear coefficient of determination. Evidence is presented that predictor gene combinations can be effectively used to resolve gene-gene interactions regulated by (Italic)Ahr(/Italic) ligands. (Italic)Key words:(/Italic) aryl hydrocarbon receptor, bioinformatics, gene networks, genomics. (Italic)Environ Health Perspect (/Italic)112:403-412 (2004). [Online 14 January 2004]

Lead (Pb) and mercury (Hg) are widespread environmental contaminants that induce prominent neural toxicity. Although the brain is not the major Pb and Hg depot in the body, these metals preferentially accumulate in astroglia to exert toxic effects. In this study, we examined the effects of Pb acetate and HgCl(2) on the expression of GRP78, a molecular chaperone in the endoplasmic reticulum (ER) that may provide cytoprotection in response to cellular stresses in the C6 rat glioma cell line. We also evaluated the DNA binding activities of several redox-regulated transcription factors in metal-treated cells. Our results showed that mRNA levels of GRP78 were up-regulated by Pb and Hg at 0.1 and 1 micro M, but down-regulated at higher concentrations (10 micro M). GRP78 protein levels increased in a concentration- and time-dependent manner in Pb and/or Hg-treated cells. Pb increased protein binding to the GST- Upsilon a antioxidant/electrophile response element (ARE/EpRE) and to the NF- kappaB consensus binding sequence of the cytomegalovirus 2 (CMB2) promoter, but decreased protein binding to the Ha-ras ARE/EpRE or to the c-fos 12-O-tetradecanoyl-phorbol-13-acetate (TPA) response element (TRE). In contrast, Hg activated DNA binding by all redox-regulated transcription factors. These studies shed some light on the molecular mechanisms of Pb and Hg toxicity in C6 rat glioma cells and suggest that GRP78 and oxidative stress may participate in the neurotoxic response to these metals.

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Atherogenic stimuli trigger complex responses in vascular smooth muscle cells (VSMCs) that culminate in activation/repression of overlapping signal transduction cascades involving oxidative stress. In the case of benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon present in tobacco smoke, the atherogenic response involves interference with redox homeostasis by oxidative intermediates of BaP metabolism. The present studies were conducted to define genomic profiles and predictive gene biological networks associated with the atherogenic response of murine (aortic) VSMCs to BaP. A combined oxidant-antioxidant treatment regimen was used to identify redox-sensitive targets during the early course of the atherogenic response. Gene expression profiles were defined using cDNA microarrays coupled to analysis of variance and several clustering methodologies. A predictor algorithm was then applied to gain insight into critical gene-gene interactions during atherogenesis. Supervised and nonsupervised analyses identified clones highly regulated by BaP, unaffected by antioxidant, and neutralized by combined chemical treatments. Lymphocyte antigen-6 complex, histocompatibility class I component factors, secreted phosphoprotein, and several interferon-inducible proteins were identified as novel redox-regulated targets of BaP. Predictor analysis confirmed these relationships and identified immune-related genes as critical molecular targets of BaP. Redox-dependent patterns of gene deregulation indicate that oxidative stress plays a prominent role during the early stages of BaP-induced atherogenesis.