Aristolochic acid and the etiology of endemic (Balkan) nephropathy

doi:10.1073/pnas.0701248104

Aristolochic acid and the etiology of endemic (Balkan) nephropathy

*Laboratory of Chemical Biology, Department of Pharmacological Sciences, and
^‡Department of Pathology, Stony Brook University, Stony Brook, NY 11794;
^¶Department of Urological Surgery and
^‖Department of Pathology, Josip Benčević General Hospital, 35000 Slavonski Brod, Croatia;
^§Roche Molecular Systems, Pleasanton, CA 94588;
**Institute Rudjer Bošković, 10000 Zagreb, Croatia;
^††Division of Environmental Disease Prevention, Wadsworth Center, New York State Department of Health, Albany, NY 12201;
^‡‡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

Edited by Arno G. Motulsky, University of Washington School of Medicine, Seattle, WA, and approved June 8, 2007 (received for review February 9, 2007)

Abstract

Endemic (Balkan) nephropathy (EN), a devastating renal disease affecting men and women living in rural areas of Bosnia, Bulgaria, Croatia, Romania, and Serbia, is characterized by its insidious onset, invariable progression to chronic renal failure and a strong association with transitional cell (urothelial) carcinoma of the upper urinary tract. Significant epidemiologic features of EN include its focal occurrence in certain villages and a familial, but not inherited, pattern of disease. Our experiments test the hypothesis that chronic dietary poisoning by aristolochic acid is responsible for EN and its associated urothelial cancer. Using ³²P-postlabeling/PAGE and authentic standards, we identified dA-aristolactam (AL) and dG-AL DNA adducts in the renal cortex of patients with EN but not in patients with other chronic renal diseases. In addition, urothelial cancer tissue was obtained from residents of endemic villages with upper urinary tract malignancies. The AmpliChip p53 microarray was then used to sequence exons 2–11 of the p53 gene where we identified 19 base substitutions. Mutations at A:T pairs accounted for 89% of all p53 mutations, with 78% of these being A:T → T:A transversions. Our experimental results, namely, that (i) DNA adducts derived from aristolochic acid (AA) are present in renal tissues of patients with documented EN, (ii) these adducts can be detected in transitional cell cancers, and (iii) A:T → T:A transversions dominate the p53 mutational spectrum in the upper urinary tract malignancies found in this population lead to the conclusion that dietary exposure to AA is a significant risk factor for EN and its attendant transitional cell cancer.

Footnotes

^†To whom correspondence should be addressed. E-mail: apg{at}pharm.stonybrook.edu
Author contributions: A.P.G., S.S., F.M., U.M., R.J.T., and B.J. designed research; L.W., N. Suzuki, A.F., T.R., Z.M., K.J., B.B., N. Slade, A.K.G., and R.R. performed research; Z.M., K.J., and M.V. contributed new reagents/analytic tools; A.P.G., S.S., M.M., F.M., L.W., K.J., R.J.T., M.V., and B.J. analyzed data; and A.P.G., R.J.T., and B.J. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0701248104/DC1.
Abbreviations:

AA,

aristolochic acid (mixture of AA-I and AA-II);

AAN,

aristolochic acid nephropathy;

AL,

aristolactam;

dA-AL-I,

7-(deoxyadenosin-N6-yl) aristolactam-I;

dA-AL-II,

7-(deoxyadenosin-N6-yl) aristolactam-II;

dG-AL,

7-(deoxyguanosin-N2-yl) aristolactam-I;

dNs,

nucleotides/nucleosides;

EN,

endemic (Balkan) nephropathy.
Freely available online through the PNAS open access option.