TP53 Mutational signature for aristolochic acid: an environmental carcinogen Masaaki Moriya 1 , Neda Slade 2 , Branko Brdar 3 , Zvonimir Medverec 4 , Karla Tomic 5 , Bojan Jelakovic ´ 6 , Lin Wu 7 , Sim Truong 7 , Andrea Fernandes 1 and Arthur P. Grollman 1 1 Laboratory of Chemical Biology, Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY 11794 2 Division of Molecular Medicine, Rudjer Bos ˇkovic Institute, 10000 Zagreb, Croatia 3 Division of Molecular Biology, Rudjer Bos ˇkovic ´ Institute, 10000 Zagreb, Croatia 4 Department of Urological Surgery, Josip Benc ˇevic ´ General Hospital, 35000 Slavonski Brod, Croatia 5 Department of Pathology, Josip Benc ˇevic ´ General Hospital, 35000 Slavonski Brod, Croatia 6 Department of Nephrology and Arterial Hypertension, Zagreb University School of Medicine and University Hospital Center, 10000 Zagreb, Croatia, and Croatian Center for Endemic Nephropathy, 35000 Slavonski Brod, Croatia 7 Roche Molecular Systems, Pleasanton, CA 94588 This study was designed to establish the TP53 mutational spectrum of aristolochic acid (AA), examined in the context of endemic (Balkan) nephropathy, an environmental disease associated with transitional cell (urothelial) carcinomas of the upper urinary tract (UUC). Tumor tissue was obtained from residents of regions in Bosnia, Croatia and Serbia where endemic nephropathy has been prevalent for over 50 years. Fifty-nine TP53 mutations were detected in 42 of the 97 tumors analyzed. Mutational spectra were dominated by A:T to T:A transversions with the mutated adenines located almost exclusively on the nontranscribed strand. This marked strand bias is attributed to selective processing of aristolactam-dA adducts by transcription-coupled nucleotide excision repair. Hotspots for A:T to T:A mutations include codons 131 and 179 and the 5 0 -AG acceptor splice site of intron 6. The unique TP53 mutational signature for AA identified in this study can be used to explore the hypothesis that botanical products containing this human carcinogen and nephrotoxin are responsible, in part, for the high prevalence of UUC and chronic renal disease in countries where Aristolochia herbal remedies traditionally have been used for medicinal purposes. Endemic (Balkan) nephropathy (EN) is a chronic renal tubu- lointerstitial disease affecting residents of rural villages situated near tributaries of the Danube River. 1 Significant epi- demiologic features of EN include its presence in certain farming villages in Bosnia and Herzegovina, Bulgaria, Croatia, Romania and Serbia; a familial, but not inherited pattern of disease and a strong association with transition cell (urothelial) carcinomas of the upper urinary tract (UUC). 2 It has long been suspected that EN is an environmental disease, and the role of environmental nephrotoxins, most notably ochratoxin A, has been investigated extensively over the past 50 years. 1 Our research, focusing on the etiology of EN/UUC, was prompted by the striking clinical and histopa- thologic similarities between EN/UUC and aristolochic acid nephropathy (AAN), the latter highlighted by a cluster of end stage renal failure cases among Belgian women who in- advertently had ingested Aristolochia herbs. 3 The hypothesis that aristolochic acid (AA) plays a causative role in the etiol- ogy of EN is supported by observations that residents of endemic villages potentially are exposed to this nephrotoxin/ carcinogen through the ingestion of AA-contaminated bread. 4,5 Direct evidence for such exposure was provided by the identification of aristolactam (AL)-DNA adducts in the renal cortex and urothelial tissues of patients with EN/UUC. 6 Somatic TP53 mutations are present in more than half of all human cancers; the mutational spectrum of this tumor suppressor gene provides clues to the etiology and molecular pathogenesis of human cancers. 7 Thus, the association of spe- cific TP53 mutations with prior exposure to environmental carcinogens gave rise to the concept of mutational ‘‘signa- tures’’. 8 For example, the high frequency of G:C to T:A trans- versions at the third base of codon 249 of TP53 was used to establish the relationship between exposure to aflatoxin B1 Key words: aristolochic acid, TP53, signature mutation, urothelial cancer, Balkan endemic nephropathy Additional Supporting Information may be found in the online version of this article. Grant sponsor: National Institute of Environmental Health Sciences (NIH); Grant number: P01ES-004068; Grant sponsor: the Croatian Ministry of Science DOI: 10.1002/ijc.26077 History: Received 17 Dec 2010; Accepted 22 Feb 2011; Online 16 March 2011 Correspondence to: Arthur P. Grollman, Laboratory of Chemical Biology, Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY 11794-8651, USA, E-mail: apg@pharm. stonybrook.edu Short Report Int. J. Cancer: 129, 1532–1536 (2011) V C 2011 UICC International Journal of Cancer IJC