Toxicogenomic analysis of aristolochic acid-induced epithelial injury and carcinogenesis

Proc Amer Assoc Cancer Res, Volume 47, 2006 1905 Aristolochic acid (AAI) is a naturally occurring herbal toxin whose cumulative ingestion results in tubulointerstitial fibrosis and leads to end-stage renal failure. Patients with aristolochic acid-associated nephropathy (AAN) also exhibit high prevalence of upper urothelial carcinomas. We used an in vivo model of AAN to explore molecular and cellular mechanisms underlying genotoxic and cytotoxic effects of AAI. The susceptible mouse strain C3H/He was treated by daily intraperitoneal administration of AAI. Accumulation of DNA adducts in the renal cortex was demonstrated by 32P-post-labeling methods. Histopathological evaluation revealed progressive cell death (necrosis/apoptosis), significant inflammation and fibrosis starting on day 3. RNA extracted from the renal cortex was subjected to DNA array analyses (7 time-points with time-matched untreated animals as controls) using Affymetrix mouse genome 430A 2.0 arrays. Rigorous statistical filtering and gene ontology (GO, biological process) representation analyses revealed extensive transcriptional reprogramming including rapid mobilization of genes involved in p53-dependent DNA damage and repair responses, cell cycle arrest and apoptosis (Cdkn1a/p21, Pcna, Gadd45b/g, Ercc1, Rad51), inflammatory/immune response (monocyte chemotactic proteins Ccl2/Mcp1 and Ccl8/Mcp2, Cxcl1/Gro1, C3), and fibrogenic mechanisms implicating TGF-β directed epithelial-mesenchymal transition (EMT) in the tubular epithelia (Tgfb1, Tgfbr1, Fos, S100a4/Fsp1, Col1a1, Col3a1, Fn1, Ctgf, Dcn, Mmp2). Next, we used Affymetrix human genome U133A 2.0 arrays to analyze primary (HKPTC) and immortalized (HK-2) human proximal tubular cells treated by AAI. We observed broad overlap between the mouse model and cultured human cells in both rapid DNA adduct formation and expression patterns involving key gene orthologs, supporting our primary hypothesis that proximal tubular cells are the primary target in AAN. In conclusion, our toxicogenomic approach provides novel insights into molecular networks underlying pathogenesis of AAN and is being extended to expression profiling of carcinogenic effects of AAI in murine urothelium.. (Research supported by NIH grant ES 04068)