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Exposure of cultured glomeruli to benzo[a]pyrene (BaP), a carcinogenic hydrocarbon, modulates mesangial and visceral epithelial cell proliferation in vivo and in vitro. The present studies were conducted to characterize mitogenic signaling profiles of cultured glomeruli following repeated cycles of BaP challenge. Enhanced rates of DNA synthesis were observed by the third passage in randomly cycling cultures after single or repeated carcinogen exposure. This response was characterized by upregulation of mitogenic sensitivity during early cell cycle transit, and increased cell numbers under restrictive growth conditions. The mitogenic response to platelet-derived growth factor (0.5 to 25 ng/mL), acidic fibroblast growth factor (2.5 to 10 ng/mL), basic fibroblast growth factor (0.05 to 5 ng/mL), epidermal growth factor (0.5 to 5 ng/mL), or conditioned medium was not enhanced by hydrocarbon challenge. BaP-treated cultures exhibited anchorage-independent growth and increased expression of hepatocyte growth factor mRNA and E-cadherin protein. Binding of activator protein-1 to DNA was enhanced in BaP-treated cells, but this change did not involve truncation or mutation of the c-jun delta region. Collectively, the data demonstrate that repeated cycles of BaP injury alter mitogenic signaling profiles in cultured glomerular cells. These alterations may contribute to deregulation of proliferative control following carcinogen exposure in vivo.

The cytotoxic responses of cultured rat renal cortico-tubular epithelial cells (TECs) and glomerular mesangial cells (GMCs) to selected polycyclic and polyhalogenated aromatic hydrocarbons (AHs) were evaluated to further define class specific differences in toxicity potential among these chemicals. Primary cultures of renal cells were exposed to benzo[ a ]pyrene (BaP, 0.3–30 μ m ); naphthalene (NAPH, 1–1000 μ m ); 2-methylnaphthalene (2-MNAPH, 1–1000 μ m ), 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD, 0.01–1n m ); 2,3,7,8-tetrachlorodibenzofuran (TCDF, 0.05–5n m ; and pentachlorophenol (PCP, 0.1–10 μ m for 4 or 24 hr. Measurements of mitochondrial membrane fragility in TECs revealed that the polycyclic aromatic hydrocarbons (PAHs) (BaP, NAPH and 2-MNAPH) preferentially injured these cells relative to halogenated aromatic hydrocarbons (HAHs). Extended exposures to all AHs tested were associated with moderate mitochondrial injury in TECs. Challenge of TECs with AHs for 4 hr did not modulate GSH levels, although modest increases in this tripeptide occurred on extended exposures. Similar response profiles were observed in GMCs, where PAHs elicited mitochondrial damage by 4 hr, while extended challenge was associated with injury in response to all AHs. Only aryl hydrocarbon receptor (AhR) ligands (BaP, TCDD and TCDF) depleted intracellular glutathione (GSH) in GMCs, while extended exposures to BaP and TCDD, as well as NAPH and 2-MNAPH, were associated with rebound increases in cellular GSH content. These data indicate that AHs can compromise renal cell function by interference with mitochondrial function and GSH homeostasis and implicate both epithelial and mesenchymal populations in the nephrotoxic response to this heterogeneous class of chemicals.

The aryl-hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates the toxicity of certain halogenated aromatic hydrocarbons including 2,3,7,8-tetra-chlorodibenzo-p-dioxin (TCDD). These compounds are potent developmental toxicants that can alter gene expression and disrupt processes of proliferation and differentiation. It has not yet been determined which tissues during development are most sensitive to these compounds, nor which genes are directly associated with the toxicities. We developed a transgenic (TG) mouse model to delineate the temporal and spatial context of transcriptionally active AhR by utilizing a dioxin responsive element-linked LacZ reporter system. The present study focuses on the pattern of TCDD-induced transgene expression localized to the footpad and digits of the paws between gestational days (GD) 13 and 18. Paw morphology was evaluated at several developmental stages following TCDD exposure. Gene expression profiles acquired by microarray technology were evaluated in the paws of fetuses exposed at GD 14.5. The results showed that TCDD exposure in utero induced LacZ expression in the developing paws. This expression appeared to be localized to the mesenchymal cell layer. Gross morphological changes were not observed in the paws prior to or after birth following TCDD exposure in utero. However, significant alterations in gene expression profiles in the developing paws were observed at 24 h following TCDD exposure in utero. These results indicate that the developing paw is a target tissue of TCDD in terms of altered gene expression, further validating the use of this AhR responsive reporter gene TG mouse model in studying AhR ligand-mediated responsiveness. However, the linkage of these changes to detectable biological outcomes in the paw remains unclear.

Phenotypic profiles of cultured glomerular cells following repeated cycles of hydrocarbon injury. Background The glomerulus has been implicated as a target of hydrocarbon injury in vitro and in vivo. In the present studies, the phenotypic profiles of cultured rat glomerular cells (GCs) following repeated cycles of hydrocarbon injury were evaluated. Cultured GCs were incubated for 24 hours with benzo[a]pyrene (BaP; 3 μmol/L), a prototypical polycyclic aromatic hydrocarbon, and were allowed to recover overnight before two additional cycles of chemical challenge during serial propagation in vitro. At the end of this regimen, control cultures were characterized by predominance of fusiform cells that grew in "hills and valleys," while GCs subjected to hydrocarbon injury displayed an epithelial morphology characterized by a rounded, polygonal shape clearly distinct from that normally exhibited by glomerular mesangial cells (GMCs) in culture. Methods Indirect immunofluorescent detection of cell markers was conducted to identify cells of mesenchymal or epithelial origin. Measurements of DNA synthesis and cell number were performed to determine proliferative capacities of the different cell types in response to hydrocarbon challenge. Results Immunofluorescence studies revealed that control GC cultures contained mostly α-smooth muscle (SM) actin-positive cells, with a few (5.1%± 2.6) E-cadherin–positive cells occasionally identified. In contrast, BaP-treated cultures exhibited a mixed cell population in which E-cadherin–positive cells were predominant (66.6%± 4.1). Single-cell cloning of naive cultures of GCs yielded four clones, three of which exhibited a fusiform morphology and were α-SM actin positive (SCC 1 through SCC 3) and one (SCC 4E) that exhibited epithelial characteristics similar to those found in hydrocarbon-treated cultures. Immunofluorescence studies showed that epithelial cells in hydrocarbon-treated cultures, as well as SCC 4E-derived clones, were vimentin positive and cytokeratin negative, characteristics similar to glomerular visceral epithelial cells (GVECs). DNA synthesis and cell proliferation in clone SCC 1 were decreased following acute BaP challenge, while growth rates in SCC 4E-derived clones were unaffected by hydrocarbon injury. Repeated cycles of hydrocarbon challenge in clonal populations yielded different profiles of DNA synthesis, with significant decreases in SCC 1 and no changes in SCC 4E. Conclusions These observations suggest that hydrocarbon injury induces differential responses in cells of the glomerulus, resulting in inhibition of GMCs and selective growth advantage of GVECs. These alterations are reminiscent of critical events described in the pathogenesis of focal segmental glomerulosclerosis and raise important questions about the pathogenesis of hydrocarbon-induced nephropathies.