Branko Brdar

Aristolochic acid and the etiology of endemic (Balkan) nephropathy

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.

Aristolactam-DNA adducts are a biomarker of environmental exposure to aristolochic acid

Endemic (Balkan) nephropathy is a chronic tubulointerstitial disease frequently accompanied by urothelial cell carcinomas of the upper urinary tract. This disorder has recently been linked to exposure to aristolochic acid, a powerful nephrotoxin and human carcinogen. Following metabolic activation, aristolochic acid reacts with genomic DNA to form aristolactam-DNA adducts that generate a unique TP53 mutational spectrum in the urothelium. The aristolactam-DNA adducts are concentrated in the renal cortex, thus serving as biomarkers of internal exposure to aristolochic acid. Here, we present molecular epidemiologic evidence relating carcinomas of the upper urinary tract to dietary exposure to aristolochic acid. DNA was extracted from the renal cortex and urothelial tumor tissue of 67 patients that underwent nephroureterectomy for carcinomas of the upper urinary tract and resided in regions of known endemic nephropathy. Ten patients from nonendemic regions with carcinomas of the upper urinary tract served as controls. Aristolactam-DNA adducts were quantified by (32)P-postlabeling, the adduct was confirmed by mass spectrometry, and TP53 mutations in tumor tissues were identified by chip sequencing. Adducts were present in 70% of the endemic cohort and in 94% of patients with specific A:T to T:A mutations in TP53. In contrast, neither aristolactam-DNA adducts nor specific mutations were detected in tissues of patients residing in nonendemic regions. Thus, in genetically susceptible individuals, dietary exposure to aristolochic acid is causally related to endemic nephropathy and carcinomas of the upper urinary tract.

TP53 Mutational signature for aristolochic acid: an environmental carcinogen

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′‐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.

p53 mutations as fingerprints for aristolochic acid: an environmental carcinogen in endemic (Balkan) nephropathy

The activation of protooncogenes and inactivation of tumor suppressor genes are considered to be the main molecular events in the multistep process of carcinogenesis. Mutations of the TP53 tumor suppressor gene have been found in nearly all tumor types and are estimated to contribute to more than 50% of all cancers. Most mutations lead to the synthesis of highly stable, inactive proteins that accumulate in the nucleus of cancer cells. Among the 393 codons of the human p53 gene, 222 are targets of 698 different types of mutations. Alterations of codons 175, 248, 273 and 282 correspond to 19% of all mutations and are considered general hot spot mutations. Dietary exposure to aristolochic acid (AA), an established nephrotoxin and human carcinogen found in all Aristolochia species was shown to be the causative agent of aristolochic acid nephropathy (previously called Chinese herbs nephropathy). This syndrome is characterized by proximal tubular damage, renal interstitial fibrosis, slow progression to the end stage renal disease and a high prevalence of upper urinary tract urothelial carcinoma (otherwise a highly unusual location). AA preferentially binds to purines in DNA and is associated with a high frequency of A-->T transversions in the p53 gene. Rats treated with AA develop A:T-->T:A mutations in codon 61. The pathological and clinical features of endemic (Balkan) nephropathy closely resemble those associated with aristolochic acid nephropathy except for the slower progression to end stage renal disease and longer cumulative period before the appearance of urothelial cancer. Recently, we reported the presence of AA-DNA adducts in renal cortex and A-->T p53 mutations in tumor tissue of patients from Croatia and Bosnia with endemic nephropathy. These data support the hypothesis that dietary exposure to AA is a major risk factor for endemic (Balkan) nephropathy.

Inhibition of viral RNA synthesis by 1-methyl-1-nitrosourea.

Abstract The effect of 1-methyl-1-nitrosourea on the synthesis of viral RNA of Poliovirus in KB-cells has been examined. It was found that 1-methyl-1-nitrosourea at concentration of 400 μg/ml blocked the synthesis of viral RNA in Poliovirus-infected KB-cells irrespective of whether it was added to culture medium immediately after virus adsorption or 2 h later. Furthermore, the inhibitory effect of 1-methyl-1-nitrosourea on the growth of Poliovirus is comparable to that of Herpes simplex virus grown in KB-cells. These findings suggest that 1-methyl-1-nitrosourea does not discriminate between these two types of viruses and moreover that the viral RNA of Poliovirus could also be the target for the action of the drug.

Abstract LB-401: TP-53 mutational signature of a urothelial carcinogen

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Aristolochic acid (AA), a powerful nephrotoxin and human carcinogen, was shown recently (PNAS, 102, 12129 (2007)) to be the causative agent in endemic (Balkan) nephropathy (EN). This devastating environmental disease is associated with a >50% incidence of urothelial carcinomas of the upper urinary tract (UUC). Following metabolic activation, AA reacts with genomic DNA to form covalent adducts (AL-DNA). These mutagenic lesions persist for decades in the renal cortex where they serve as robust biomarkers of exposure to AA. In EN, exposure occurs through ingestion of bread prepared from flour contaminated with AA. In China and other Asian countries, a significant fraction of the population also is exposed to AA, in this case through the medicinal use of herbal medicines prepared fr om Aristohchia plants. Such exposure, well-documented in Taiwan (JNCI, 102 1 (2009)) raises an important question: Do AA-induced cancers occur globally as a silent iatrogenic disease? To address this public health issue, we undertook a systematic study of 75 UUC patients residing in regions of Croatia, Bosnia and Serbia where EN and UUC are prevalent. DNA was obtained, with informed consent, from renal cortex and tumor tissues following nephroureterectomy. AL-DNA adducts were quantified by 32P- post-labeling techniques. Chip-sequencing technology was utilized to detect base substitutions in the TP-53 gene. Adducts were found in the renal cortex of 65% of patients with UUC. The TP53 mutation spectrum was dominated by A: TaT: A transversions located almost exclusively on the non-transcribed DNA strand. Twenty-five of these mutations occurred in exons and 12 mutations at 5′AG splice sites. Additionally, we identified several A: T aT: A mutational hotspots, including the first base of codon 131. TP53 gene mutations at this position have not previously been reported. The mechanism underlying the marked strand bias appears to be a selective failure to excise AL-DNA adducts by global genomic nucleotide excision repair. This factor also may account for the remarkable persistence of these adducts in human tissues, in some cases, for more than 50 years. Thus, AA joins vinyl chloride and aflatoxin as human chemical carcinogens with a definitive mutational signature. This information, coupled with the use of AL-DNA adducts as a biomarker, may be used to establish the role of AA ingestion in countries with a high prevalence of UUC and chronic renal disease. Importantly, public health authorities in countries where Aristolochia herbal preparations are in use should implement measures to reduce human exposure to this toxic and carcinogenic herb. (Supported by grant ES-04068 from NIEHS and the Croatian Ministry of Science) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-401.