Robert J. Turesky

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.

Aristolochic acid-associated urothelial cancer in Taiwan

Aristolochic acid, a potent human carcinogen produced by Aristolochia plants, is associated with urothelial carcinoma of the upper urinary tract (UUC). Following metabolic activation, aristolochic acid reacts with DNA to form aristolactam (AL)-DNA adducts. These lesions concentrate in the renal cortex, where they serve as a sensitive and specific biomarker of exposure, and are found also in the urothelium, where they give rise to a unique mutational signature in the TP53 tumor-suppressor gene. Using AL-DNA adducts and TP53 mutation spectra as biomarkers, we conducted a molecular epidemiologic study of UUC in Taiwan, where the incidence of UUC is the highest reported anywhere in the world and where Aristolochia herbal remedies have been used extensively for many years. Our study involves 151 UUC patients, with 25 patients with renal cell carcinomas serving as a control group. The TP53 mutational signature in patients with UUC, dominated by otherwise rare A:T to T:A transversions, is identical to that observed in UUC associated with Balkan endemic nephropathy, an environmental disease. Prominent TP53 mutational hotspots include the adenine bases of 5′AG (acceptor) splice sites located almost exclusively on the nontranscribed strand. A:T to T:A mutations also were detected at activating positions in the FGFR3 and HRAS oncogenes. AL-DNA adducts were present in the renal cortex of 83% of patients with A:T to T:A mutations in TP53, FGFR3, or HRAS. We conclude that exposure to aristolochic acid contributes significantly to the incidence of UUC in Taiwan, a finding with significant implications for global public health.

Metabolism and Biomarkers of Heterocyclic Aromatic Amines in Molecular Epidemiology Studies: Lessons Learned from Aromatic Amines

Aromatic amines and heterocyclic aromatic amines (HAAs) are structurally related classes of carcinogens that are formed during the combustion of tobacco or during the high-temperature cooking of meats. Both classes of procarcinogens undergo metabolic activation by N-hydroxylation of the exocyclic amine group to produce a common proposed intermediate, the arylnitrenium ion, which is the critical metabolite implicated in toxicity and DNA damage. However, the biochemistry and chemical properties of these compounds are distinct, and different biomarkers of aromatic amines and HAAs have been developed for human biomonitoring studies. Hemoglobin adducts have been extensively used as biomarkers to monitor occupational and environmental exposures to a number of aromatic amines; however, HAAs do not form hemoglobin adducts at appreciable levels, and other biomarkers have been sought. A number of epidemiologic studies that have investigated dietary consumption of well-done meat in relation to various tumor sites reported a positive association between cancer risk and well-done meat consumption, although some studies have shown no associations between well-done meat and cancer risk. A major limiting factor in most epidemiological studies is the uncertainty in quantitative estimates of chronic exposure to HAAs, and thus, the association of HAAs formed in cooked meat and cancer risk has been difficult to establish. There is a critical need to establish long-term biomarkers of HAAs that can be implemented in molecular epidemioIogy studies. In this review, we highlight and contrast the biochemistry of several prototypical carcinogenic aromatic amines and HAAs to which humans are chronically exposed. The biochemical properties and the impact of polymorphisms of the major xenobiotic-metabolizing enzymes on the biological effects of these chemicals are examined. Lastly, the analytical approaches that have been successfully employed to biomonitor aromatic amines and HAAs, and emerging biomarkers of HAAs that may be implemented in molecular epidemiology studies are discussed.

Mutational Signature of Aristolochic Acid Exposure as Revealed by Whole-Exome Sequencing

The mutational signature of aristolochic acid exemplifies how genome-wide sequencing can be used to identify environmental exposures leading to cancer. Carcinogen AAlert Aristolochic acid (AA) is a natural compound derived from plants in the Aristolochia genus. For centuries, Aristolochia has been used throughout Asia to treat a variety of ailments as a component of traditional Chinese medicine. In recent years, however, a more sinister side of this herb has come to light when it was linked to kidney damage and cancers of the urinary tract. Now, two studies by Poon et al. and Hoang et al. present a “molecular signature” of AA-induced DNA damage, which helps to explain the mutagenic effects of AA and may also be useful as a way to detect unsuspected AA exposure as a cause of cancer. The molecular signature seen in AA-associated tumors is characterized by a predominance of A:T-to-T:A transversions, a relatively unusual type of mutation that is infrequently seen in other types of cancer, including those caused by other carcinogens. These mutations concentrate at splice sites, causing the inappropriate inclusion or exclusion of entire exons in the resulting mRNA. The overall mutation rate is another notable feature of AA-associated cancers, because it is several times higher than the rate of mutations caused by other carcinogens such as tobacco and ultraviolet light. In both studies, the authors also used the molecular signature to discover that AA was a likely cause of tumors previously attributed to other carcinogens. In one case, a urinary tract cancer that had been attributed to smoking and, in the other case, a liver cancer previously attributed to a chronic hepatitis infection were both identified as having the telltale signature of AA mutagenesis. The identification of a specific molecular signature for AA has both clinical and public health implications. For individual patients, the molecular signature could help physicians identify which tumors were caused by AA. Although this information cannot yet be used to optimize the treatment of individual patients, those who are diagnosed with AA-associated cancers could be monitored more closely for the appearance of additional tumors. Meanwhile, a better understanding of the mutagenic effects of AA should also help to strengthen public health efforts to decrease exposure to this carcinogenic herb. In humans, exposure to aristolochic acid (AA) is associated with urothelial carcinoma of the upper urinary tract (UTUC). Exome sequencing of UTUCs from 19 individuals with documented exposure to AA revealed a remarkably large number of somatic mutations and an unusual mutational signature attributable to AA. Most of the mutations (72%) in these tumors were A:T-to-T:A transversions, located predominantly on the nontranscribed strand, with a strong preference for deoxyadenosine in a consensus sequence (T/CAG). This trinucleotide motif overlaps the canonical splice acceptor site, possibly accounting for the excess of splice site mutations observed in these tumors. The AA mutational fingerprint was found frequently in oncogenes and tumor suppressor genes in AA-associated UTUC. The AA mutational signature was observed in one patient’s tumor from a UTUC cohort without previous indication of AA exposure. Together, these results directly link an established environmental mutagen to cancer through genome-wide sequencing and highlight its power to reveal individual exposure to carcinogens.

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.

Analysis of mutagenic heterocyclic amines in cooked beef products by high-performance liquid chromatography in combination with mass spectrometry

Mutagenic activity detected in beef extracts and in fried beef heated for varying periods of time was purified and then analysed by high-performance liquid chromatography in combination with mass spectrometry (LC-MS). The major mutagenic component found in all of the beef products was identified as 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) followed by lesser amounts of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx). Identification and quantification of mutagens were achieved by the use of deuterium-labelled analogues. Measured levels of MeIQx and 4,8-DiMeIQx in different batches of beef extract were in the range 11.7-52.2 and 0-11.2 ng/g, respectively, and in beef heated at 275 degrees C for 5-15 min the values of MeIQx and 4,8-DiMeIQx were in the range 2.7-12.3 and 0-3.9 ng/g, respectively. The levels of IQ found in beef extracts were 0-36.8 ng/g and in fried beef the amounts were estimated at 0.3-1.9 ng/g. The method of purification is rapid, requiring only XAD-2 adsorption followed by an acid-base liquid partition against ethyl acetate and blue cotton treatment (trisulpho-copper-phthalocyanine) prior to LC-MS analysis. Because of the sensitivity of LC-MS, mutagens present in cooked beef can be detected at the low parts-per-billion-level and as little as 10 g of cooked beef was required for analysis.

HETEROCYCLIC AROMATIC AMINE METABOLISM, DNA ADDUCT FORMATION, MUTAGENESIS, AND CARCINOGENESIS

Heterocyclic aromatic amines (HAAs) are carcinogenic compounds formed in meats, fish, and poultry prepared under common household cooking practices. Some HAAs are also formed in tobacco smoke condensate. Because of the widespread occurrence of HAAs in these daily staples, health concerns have been raised regarding the potential role of HAAs in the etiology of some human cancers associated with frequent consumption of these products. In this review, the metabolism of HAAs to biologically active metabolites that bind to DNA and provoke mutations and cancer in various biological systems is discussed. Some of the current analytical and molecular methods that are used to measure biomarkers of HAA exposure and genetic damage in experimental animal models and humans are also presented. These biochemical data combined may help to better assess the role that HAAs may have in the development of some common forms of human cancers.

The coffee components kahweol and cafestol induce γ-glutamylcysteine synthetase, the rate limiting enzyme of chemoprotective glutathione synthesis, in several organs of the rat

Abstract. The coffee components kahweol and cafestol (K/C) were reported to be protective against mutagenic damage by heterocylic amines and aflatoxin B1 in the rat, while in humans the consumption of coffee with a high K/C content was associated with a lower rate of colon tumors. An important mechanism of this antimutagenic effect appears to be the potential of K/C to induce glutathione-S-transferase (GST) and to enhance hepatic levels of glutathione (GSH), the co-factor of GST, which is independently involved in further protective mechanisms. In the present study, we investigated mechanisms and organ specificities (liver, kidney, lung, colon) of the K/C effect on GSH levels, and particularly the role of γ-glutamylcysteine synthetase (GCS), the rate limiting enzyme of GSH synthesis. Chows containing one of four concentrations of either a 1:1 mixture of K/C (0.012–0.122%) or of cafestol alone (0.006–0.061%) were fed to male F344 rats for 10 days. In the K/C-treated livers, a dose-dependent increase of up to 2.4-fold in the activity of GCS was observed, being statistically significant even at the lowest dose, and associated with an increase in GSH of up to three-fold. Notably, the highest dose doubled the hepatic mRNAs of the heavy and light subunits of GCS, suggesting enhanced transcription. In the extrahepatic organs, GCS activity and GSH levels were increased as well, although more moderately than in the liver. Since enhancement of GCS had also been observed as a consequence of oxidative stress, the possibility of such an involvement in the actions of K/C was examined by determining hepatic thiobarbituric acid reactive substances and the ratio of oxidized and reduced GSH. However, no evidence of oxidative stress was detected. In summary, K/C increased GSH levels apparently through the induction of the rate limiting enzyme of GSH synthesis, which may be a key factor in the chemopreventive potential of coffee components.

Comparison of analytical techniques to quantify malondialdehyde in milk powders

Several analytical methods were compared to quantify malondialdehyde (MDA) in milk powders. Modified thiobarbituric acid (TBA) methods, using either visible spectrophotometry (direct absorbance reading or after third derivative transformation of the spectrum) or HPLC, required derivatisation at elevated temperature, which appeared to catalyse artefactual MDA formation and thus overestimate the MDA content. In contrast to the TBA derivatisation method, the measurement of MDA as the dinitrophenylhydrazone derivative by HPLC or as the phenylhydrazone product by GC-MS with a deuterated internal standard resulted in lower estimates in the ranges of 2-17- and 3-30-fold, respectively; apparently due to the milder derivatisation conditions. The estimates of MDA determined by both HPLC-UV and GC-MS techniques result in lower values which are similar in magnitude even though the GC-MS technique is more sensitive.

The inhibitory effects of coffee on radical-mediated oxidation and mutagenicity

Hydrogen peroxide (H2O2) has been implicated as a major contributor to coffee mutagenicity and genotoxicity in vitro. We have used three assays to show the gradual formation of H2O2 in freshly prepared roasted ground coffee and in instant coffees over time reaching levels of 400-450 microM after a 1-h incubation period. Formation of H2O2 occurs through an auto-oxidation process where polyphenolics, in the presence of transition metals, reduce atmospheric oxygen. However, because of these polyphenolics, coffee also possesses in vitro antioxidant activity as shown by its capacity to inhibit lipid peroxidation in Fenton-catalysed hydroxylation reactions. The pro- and antioxidative effects of coffee are also reflected in its mutagenic and antimutagenic activity in the Ames test. Coffee is directly mutagenic in strains TA100 and TA102 due to H2O2 formation. However, coffee is also an antioxidant and antimutagen. This beverage exerts a strong protective effect against the mutagenicity and cytotoxicity induced by the oxidant t-butylhydroperoxide (t-BOOH). Thus, coffee, like many antioxidants, exhibits dual effects in vitro which are highly dependent upon parameters such as dose, atmospheric oxygen, transition metals as well as the biological and chemical endpoints used for measurement. Consequently, the data obtained on the pro- and antioxidant properties of foods and beverages from in vitro bioassays must be interpreted with caution and the results are not easily extrapolated in vivo to assess the impact on human health.