AbstractExposure to aristolochic acid (AA) is associated with human nephropathy and urothelial cancer. The tumour suppressorTP53is a critical gene in carcinogenesis and frequently mutated in AA-induced urothelial tumours. We investigated the impact of p53 on AAI-induced nephrotoxicity and DNA damage in vivo by treatingTrp53(+/+),Trp53(+/−) andTrp53(−/−) mice with 3.5 mg/kg body weight (bw) AAI daily for 2 or 6 days. Renal histopathology showed a gradient of intensity in proximal tubular injury fromTrp53(+/+) toTrp53(−/−) mice, especially after 6 days. The observed renal injury was supported by nuclear magnetic resonance (NMR)-based metabonomic measurements, where a consistentTrp53genotype-dependent trend was observed for urinary metabolites that indicate aminoaciduria (i.e. alanine), lactic aciduria (i.e. lactate) and glycosuria (i.e. glucose). However,Trp53genotype had no impact on AAI-DNA adduct levels, as measured by³²P-postlabelling, in either target (kidney and bladder) or non-target (liver) tissues, indicating that the underlying mechanisms of p53-related AAI-induced nephrotoxicity cannot be explained by differences in AAI genotoxicity. Performing gas chromatography–mass spectrometry (GC–MS) on kidney tissues showed metabolic pathways affected by AAI treatment, but againTrp53status did not clearly impact on such metabolic profiles. We also cultured primary mouse embryonic fibroblasts (MEFs) derived fromTrp53(+/+),Trp53(+/−) andTrp53(−/−) mice and exposed them to AAI in vitro (50 µM for up to 48 h). We found thatTrp53genotype impacted on the expression of NAD(P)H:quinone oxidoreductase (Nqo1), a key enzyme involved in AAI bioactivation. Nqo1 induction was highest inTrp53(+/+) MEFs and lowest inTrp53(−/−) MEFs; and it correlated with AAI-DNA adduct formation, with lowest adduct levels being observed in AAI-exposedTrp53(−/−) MEFs. Overall, our results clearly demonstrate that p53 status impacts on AAI-induced renal injury, but the underlying mechanism(s) involved remain to be further explored. Despite the impact of p53 on AAI bioactivation and DNA damage in vitro, such effects were not observed in vivo.