Aristolochic acid activates ras genes in rat tumors at deoxyadenosine residues.

Aristolochic acid I (AAI), a nitrophenanthrene derivative, is the major component of the carcinogenic plant extract aristolochic acid, which has been used as a medicine since antiquity. Long term oral administration of AAI to male Wistar rats induces multiple tumors, mainly in the forestomach, ear duct, and small intestine. The presence of activated transforming genes was investigated in various tumors of 18 AAI treated rats, namely in 14 squamous cell carcinomas of the forestomach, 7 squamous cell carcinomas of the ear duct, 8 tumors of the small intestine, 3 tumors of the pancreas, 1 adenocarcinoma of the kidney, 1 lymphoma, and 2 metastases in the lung and the pancreas. By utilizing the tumorigenicity assay and Southern blot analysis, we have detected an activated c-Ha-ras gene in the DNAs of 5 of 5 squamous cell carcinomas of the forestomach. Direct sequencing of amplified material revealed an AT----TA transversion mutation at the second position of codon 61 of the c-Ha-ras gene (CAA to CTA) in all transfectants as well as in the 5 original rat tumors. Enzymatic amplification of ras sequences followed by selective oligonucleotide hybridization detected identical mutations in 93% (13 of 14) of forestomach tumors, in 100% (7 of 7) of ear duct tumors, and in the lung metastasis. Among those tumors tested, we had 4 cases in which the forestomach tumors and the ear duct tumors originated from the same rat, showing the same mutation in both tissues. Moreover, similar mutations were demonstrated at c-Ki-ras codon 61 in 1 of 7 ear duct tumors (CAA to CAT) and in 1 of 8 tumors of the small intestine (CAA to CTA) as well as at c-N-ras 61 (CAA to CTA) in a pancreatic metastasis. Additional transfection experiments of some tumors scoring negative for ras gene mutations in dot blot analyses revealed a CAA to CTA transversion at codon 61 of the c-Ha-ras gene in 1 forestomach tumor as well as at codon 61 of the c-N-ras in 1 hyperplasia of the pancreas and in 1 lymphoma. The apparent selectivity for mutations at adenine residues in AAI induced tumors is consistent with the identification of an N6-deoxyadenosine-AAI adduct formed by reaction of AAI with DNA in vitro, suggesting that carcinogen-deoxyadenosine adducts are the critical lesions in the tumor initiation by aristolochic acid.

DNA adducts in human breast tissue: association with N-acetyltransferase-2 (NAT2) and NAT1 genotypes.

The etiology of human breast cancer is poorly understood, but circumstantial evidence points toward exogenous genotoxins as causative agents; they are believed to exert their carcinogenic action by binding to DNA. Because this binding is often preceded by metabolic activation, it is dependent on the expression and activities of metabolic enzymes of the host. Human mammary tissue samples from 42 women undergoing surgery for breast cancer or reduction mammoplasty were analyzed for DNA adducts by 32P-postlabeling analysis. With the butanol extraction method of DNA adduct enrichment, adduct levels were determined to be 0-414.6 adducts per 10(9) nucleotides, with considerable interindividual variation. To characterize the DNA adducts, we reanalyzed the adduct spots by reversed-phase high-performance liquid chromatography. Of two major adduct spots detected on TLC that accounted for up to 70% of the DNA modification, one eluted as a single peak on high-performance liquid chromatography, whereas the other was resolved into two distinct peaks of radioactivity. These major adducts were highly lipophilic in character. The N-acetyltransferase-1 (NAT1) and NAT2 genes were analyzed for common mutations using random RFLP analysis. An association between NAT2 acetylator status and adduct levels was observed; significantly elevated adduct levels occurred in the mammary DNA from women who were designated slow acetylators for NAT2 [median adduct level = 83.0 adducts per 10(9) nucleotides (range, 9.0-414.6)], as compared with the levels in individuals designated rapid acetylators for NAT2 [median adduct level = 39.7 adducts per 10(9) nucleotides (range, 0-91.0; P = 0.0053)]. On the other hand, NAT1 genotypes were not significantly associated with adduct levels. Although the agents responsible for the DNA modifications in the human breast are not known, this pilot study supports the hypothesis that DNA adduct formation in the human breast may be influenced by the NAT2 genotype.

HPLC separation of 32P-postlabelled benzo[b]fluoranthene-DNA adducts.

Analysis using 32P-postlabelling and a recently developed HPLC method resolved the adduct formed by reaction of the benzo[b]fluoranthene (BbF) anti-bay-region diol-epoxide with DNA from the more polar major adduct produced by the hydrocarbon in three different biological systems. In each case, the adduct formed from the anti-bay-region diol-epoxide constituted only a minor proportion of the total DNA modification. Comparisons of the DNA adducts formed from the hydrocarbon with those formed in microsomal incubations from the putative metabolites BbF-9,10-diol, anti-BbF-9,10-diol-11,12-oxide and the 5,9,10- and 6,9,10-BbF-triols indicate that the predominant pathway for BbF activation in skin probably involves a bay-region triol-epoxide possessing a phenolic OH-group on the peninsula ring.

The structural basis for the mutagenicity of aristolochic acid.

Molecular orbital calculations with aristolochic acid I (AAI) and the model compounds 8-nitro-1-naphthoic acid (1,8NNA) and 3-nitro-2-naphthoic acid (2,3NNA) confirm a similar conformation of the nitro and carboxyl groups in these molecules. The ortho isomer 2,3NNA is not mutagenic in the Salmonella strains TA 100 or TA 1537, but the peri-substituted 1,8NNA shows mutagenic activity similar to AAI in TA 100, although it is only weakly active in TA 1537. We propose a mechanism of activation via a cyclic nitrenium ion with an aristolactam structure which is possible only in peri-substituted nitro carboxylic acids.

Cell transformation in vitro by food-derived heterocyclic amines Trp-P-1, Trp-P-2 and N(2)-OH-PhIP.

Heterocyclic aromatic amines (HCA) are formed upon frying of poultry, fish or meat and have been shown to induce tumours in rodent bioassays. We investigated the transforming activity of HCA in an in vitro assay using the M2/C3H mouse fibroblast cell line. An external metabolic activation system (rat liver homogenate) was required in order to observe any HCA-induced cytotoxic effects or cell transforming activity. Trp-P-1 and Trp-P-2 are shown to be among the most potent transforming HCA that have been detected in food. Metabolic activation of HCA has been shown to proceed via N-hydroxylation of the exocyclic amino group. Therefore, we tested 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N(2)-OH-PhIP) the activated metabolite of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. N(2)-OH-PhIP proved to be one of the most powerful compounds with transforming activity observable at a concentration as low as 30 nM. Since 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most abundant HCA formed in fried and grilled food and N-hydroxylation appears to be the predominant pathway of human metabolism, these data support the hypothesis that HCA are involved in the aetiology of human cancer.

Exposure to beta-carbolines norharman and harman.

The aromatic beta-carbolines norharman and harman have been implicated in a number of human diseases including Parkinson's disease, tremor, addiction and cancer. It has been shown that these compounds are normal body constituents formed endogenously but external sources have been identified. Here, we summarise literature data on levels of norharman and harman in fried meat and fish, meat extracts, alcoholic drinks, and coffee brews. Other sources include edible and medicinal plants but tobacco smoke has been identified as a major source. Exposure levels from these different dietary sources are estimated to a maximum of 4 microg norharman per kg body weight (bw) per day and 1 microg harman per kg bw per day. Exposure via tobacco smoke depends on smoking habits and type of cigarettes but can be estimated to 1.1 microg/kg bw for norharman and 0.6 microg/kg bw for harman per package of cigarettes smoked. Studies on toxicokinetics indicate that inhalative exposure leads to a rapid increase in plasma levels and high bioavailability of norharman and harman. Oral bioavailability is lower but there are indications that sublingual absorption may increase dietary uptake of beta-carbolines. Endogenous formation can be estimated to be 50-100 ng/kg bw per day for norharman and about 20 ng/kg bw per day for harman but these rates may increase with high intake of precursors. Biomarker studies on plasma levels of beta-carbolines reported on elevated levels of norharman, harman or both in diseased patients, alcoholics and following tobacco smoking or consumption of beta-carboline-containing food. Cigarette smoking has been identified as major influence but dietary exposure may contribute to exposure.

Investigation of the genotoxic effects of 2-amino-9H-pyrido[2,3-b]indole in different organs of rodents and in human derived cells.

Aim of the present study was the investigation of the genotoxicity of amino-alpha-carboline (AalphaC) in human derived cells and of its organ-specific effects in laboratory rodents. This heterocyclic amine (HA) is contained in fried meat and fish in higher concentrations than most other cooked food mutagens. In the present experiments, AalphaC caused dose-dependent induction of micronuclei in the human derived hepatoma cell line HepG2 at concentrations > or =50 microM. In contrast, no significant effects were seen in Hep3B, another human hepatoma cell line, which may be explained by the concurrent lower activity of sulfotransferase (SULT), an enzyme playing a key role in the activation of AalphaC. A positive result was also obtained in the single cell gel electrophoresis (SCGE) assay in peripheral human lymphocytes, but the effect was only significant at the highest concentration (1000 microM). In Fischer F344 rats and ICR mice, the liver was the main target organ for the formation of DNA adducts (at > or =50 mg/kg bw), and in lungs and colon substantially lower levels were detected. Identical organ specificity as in the DNA adduct measurements was seen in SCGE assays with rats, whereas in mice the most pronounced induction of DNA migration was observed in the colon. Comparison of our results with data from earlier experiments indicate that the genotoxic potency of AalphaC is equal to that of other HAs, which are contained in human foods in much smaller amounts. Therefore, our findings can be taken as an indication that the human health risk caused by exposure to AalphaC is higher than that of other HAs that are formed during the cooking of meat and fish.

HPLC separation of 32P-postlabelled DNA adducts formed from dibenz[a,h]anthracene in skin.

Mouse skin and human skin have been treated in vivo or in short-term organ culture with dibenz[a,h]anthracene (DB[a,h]A), the related 3,4- or 5,6-diols or the anti- or syn-3,4-diol 1,2-oxides. DNA hydrolysates have been 32P-postlabelled and the adducts present examined by HPLC using a phenyl-modified reverse phase column and, for comparison, by PEI-cellulose TLC and autoradiography. The adducts formed when the diol-epoxides were reacted with salmon sperm DNA were also examined. The results show that in mouse skin treated in vivo, the major adducts formed from DB[a,h]A and the 3,4-diol were the same and that two of them were more polar than those formed in skin or in DNA that had been treated with the related anti- or syn-diol epoxides. Human skin treated with DB[a,h]A in culture yielded an adduct profile that was qualitatively similar to the profiles obtained with mouse skin.

Pancreatic DNA adducts formed in vitro and in vivo by the food mutagens 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeA alphaC).

Genotoxic heterocyclic amines have been detected in grilled or fried meat and tobacco smoke. Among these, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeA alphaC) have been shown to induce tumours in rodents in several organs. Here we report on the DNA adduct formation by PhIP and MeA alphaC in vitro and in vivo, both in rat hepatic and rat pancreatic tissues or cells. Using 32P-postlabelling analysis both compounds were shown to induce a dose-dependent DNA modification in primary rat hepatocytes that was correlated with cytotoxicity in these cells. In explanted rat pancreas maintained in dynamic short-term organ culture MeA alphaC was shown to induce covalent DNA adducts. No DNA adducts were observed with PhIP in this assay. DNA adducts were observed in the liver and the pancreas of F344 rats treated with PhIP, with a 36-times higher level of adducts in the pancreas, confirming data reported earlier. DNA adduct levels induced by feeding 32, 160 or 800 ppm MeA alphaC in the diet were dose-dependent and higher in the liver compared with other organs including pancreas. While for PhIP the N2-(desoxyguanin-8-yl)-derivative was accounting for more than 90% of DNA adducts detected, in the case of MeA alphaC the N2-(desoxyguanin-8-yl) adduct was predominant in vitro and determined in vivo as one of up to 5 DNA adducts. MeA alphaC had been reported to induce preneoplastic foci and tumours in the liver and tumours and atrophy in the pancreas. In the case of MeA alphaC, the DNA adduct formation and cytotoxicity observed by us in vitro and in vivo correlate with the organ specificity of the reported pathological lesions. In the case of PhIP our in vitro data in pancreas and liver and the low adduct levels in liver in vivo also reflect the reported lack of pathological effects in these organs. In contrast, in pancreas, in vivo extraordinarily high adduct levels induced by PhIP were observed confirming studies published earlier, in spite of the fact that this compound does not cause pancreatic lesions. This enigmatic observation is discussed and the relevant literature is reviewed.

Morphological transformation of C3H/M2 mouse fibroblasts by, and genotoxicity of, extracts of human milk.

Breast cancer may be initiated by environmental/dietary agents and human milk may act as an ex vivo indicator of in vivo exposure of mammary epithelial cells to genotoxins. Extracts of human milk from UK-resident women (n=7) were tested for their abilities to morphologically transform C3H/M2 mouse fibroblasts. Genotoxicities were assessed in the Salmonella typhimurium reverse-mutation assay in the presence of S9 using strains TA1538 and YG1019, and in metabolically-competent human MCL-5 cells with the micronucleus and with the alkaline single cell gel electrophoresis (comet) assays. Two of the seven extracts were inactive in the transformation assay both in the presence or absence of S9, two appeared to be equally transforming either in the presence or absence of S9, and two other extracts induced increased transformation frequencies in the presence of S9. A seventh extract, tested only in the absence of S9, was inactive. Extracts were either active or inactive in at least three of the four tests applied. Four extracts were active or inactive in all four tests. The results suggest that human milk could be used as a resource for investigations of the as-yet-unidentified transforming agents previously detected in mammary lipid.

Bioactivation of the food mutagen 2-amino-3-methyl-imidazo[4, 5-f]quinoline (IQ) by prostaglandin-H synthase and by monooxygenases: DNA adduct analysis.

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) is a known multisite carcinogen in rodents and a potent mutagen in acetyltransferase-proficient Salmonella typhimurium strains on activation by either monooxygenases (MFO) or by prostaglandin H synthase (PHS). The primary metabolites formed by MFO- or PHS-mediated IQ-oxidation are different ([Wolz]), but secondary metabolism could ultimately result in the same DNA-binding intermediates. For further investigations, the DNA adduct pattern was now studied by means of (32)P-postlabelling analysis in vitro on PHS-activation and compared to that formed on MFO-mediated activation of IQ in hepatocytes. The C8-dG-IQ-adduct N-(deoxyguanosin-8-yl)-IQ was the major adduct in all samples, that is, in DNA isolated from S. typhimurium YG1024 treated with PHS-oxidized IQ or its nitro-derivative, from ovine seminal vesicle cells, and from hepatocytes exposed to IQ or nitro-IQ. This speaks for the formation of a common DNA-reactive species, presumably an arylnitrenium ion, generated by different pathways in these cellular model systems. The similarity of critical biochemical DNA lesions suggests that PHS can contribute to the bioactivation of IQ in vivo: this is of particular interest in extrahepatic tissues since expression of cytochrome P450 isoenzymes known to be involved in the N-oxidation of IQ is largely confined to the liver.

DNA adducts in marine and freshwater fish as biomarkers of environmental contamination.

The analysis of DNA modifications in aquatic animals may serve as a sensitive marker of exposure to genotoxic contaminants. This is of importance in assessing water quality regarding pollution with genotoxic compounds, food safety analysing edible aquatic animals and in terms of ecotoxicology. Covalent modification of DNA is considered a crucial event in chemical carcinogenesis and thus may be considered a biomarker of an early genotoxic effect. Measuring DNA adducts is unique in that these lesions may be considered a biomarker of both exposure and effect. A number of studies have described the analysis of the DNA isolated from the liver of both freshwater and marine fish. Considerable levels of DNA adducts have been observed in some animals from contaminated lakes or rivers. Low levels were observed in DNA from the liver of marine fish. The background levels of DNA adducts have to be determined in animals from appropriate uncontaminated control sites that are matched for species, gender, age and season of the year. It is of crucial importance to consider the species analysed since there have been reports of the non responsiveness of some species.

Improved reversed-phase high-performance liquid chromatographic separation of 32P-labelled nucleoside 3',5'-bisphosphate adducts of polycyclic aromatic hydrocarbons.

32P-Postlabelling is a sensitive technique for the detection and analysis of carcinogen-DNA adducts. In this paper we describe the development of an improved high-performance liquid chromatography (HPLC) method for the separation of 32P-labelled 3',5'-bisphosphates of nucleosides modified by reactive derivatives of carcinogenic polycyclic aromatic hydrocarbons (PAH). Optimal resolution of the major 32P-postlabelled DNA adducts formed by the anti-diol-epoxides of ten PAH was achieved using a phenyl-modified silica gel column with a gradient of methanol in phosphate buffer at low pH and high ionic strength. Use of a radioactivity flow detector coupled to the HPLC apparatus allowed detection of subfemtomole quantities of labelled adducts.

Carcinogenicity and DNA adduct formation observed in ACI rats after long-term treatment with madder root, Rubia tinctorum L.

Madder root, Rubia tinctorum L., is a traditional herbal medicine used against kidney stones. Recently we reported that lucidin, a hydroxyanthraquinone derivative present in this plant, is mutagenic in bacteria and mammalian cells. We also demonstrated the formation of DNA adducts in tissue culture and mice after treatment with this compound. To elucidate the possible carcinogenicity of madder root, three groups of male and female ACI rats received either a normal diet or a diet supplemented with 1 or 10% drug for a total period of 780 days. Weight gain and morbidity were not different among the three groups. Non-neoplastic lesions related to the treatment were evident in the liver and kidneys of both sexes. Moreover, dose-dependent increases in benign and malignant tumour formation were observed in the liver and kidneys of treated animals. 32P-post-labelling analysis showed an increase in the overall level of DNA adducts observed in the liver, kidney and colon of rats treated with 10% madder root in the diet for 2 weeks. HPLC analysis of 32P-labelled DNA adducts revealed a peak co-migrating with an adduct obtained after in vitro treatment of deoxyguanosine-3'-phosphate with lucidin. These observations suggest that the use of madder root for medicinal purposes is associated with a carcinogenic risk.

N6-adenyl arylation of DNA by aristolochic acid II and a synthetic model for the putative proximate carcinogen.

Aristolochic acid II (AAII), one of the major components of the carcinogenic plant extract aristolochic acid, is known to be mutagenic and to form DNA adducts in vitro and in vivo. The major fluorescent DNA adduct formed upon xanthine oxidase mediated reduction in the presence of calf thymus (CT-) DNA or deoxyadenosine was isolated by means of preparative HPLC and identified by fluorescence, UV/vis absorbance, and 1H NMR spectroscopy as 7-(deoxy-adenosin-N6-yl)aristolactam II. As a model proximate carcinogen, N-chloroaristolactam II was prepared chemically from aristolactam II, the reduction product of AAII. This model compound was spectroscopically characterized and found to react directly with CT-DNA without any activation, forming the same deoxyadenosine adduct. HPLC analysis with fluorescence monitoring detected this adduct in vivo in the liver DNA of Wistar rats treated orally with AAII. These results confirm the anticipated metabolic activation mechanism of AAII as occurring via a cyclic nitrenium ion.

32P-postlabelling analysis of the DNA adducts formed by aristolochic acid I and II.

We report the quantitation of DNA adducts in target and nontarget organs of male Wistar rats treated orally with five daily doses (10 mg/kg body wt) aristolochic acid I (AAI) or aristolochic acid II (AAII), the major components of the herbal drug aristolochic acid, a forestomach carcinogen in the rat. DNA adducts were detected and analysed using the nuclease P1-enhanced variation of the Randerath 32P-postlabelling assay. The highest level of DNA adducts formed was by AAI in the target organ, forestomach (330 +/- 30 adducts/10(8) nucleotides), but high levels were also observed in a non-target tissue, the glandular stomach (180 +/- 15). Lower amounts of adducts were detected in liver, kidney and urinary bladder epithelium. With AAII the binding levels were generally lower than the AAI, the highest level of adducts being detected in kidney (80 +/- 20 adducts/10(8) nucleotides) and lower levels in liver, stomach and urinary bladder epithelia. Adduct patterns similar to those in vivo were observed in two new in vitro assays. Rat faecal bacteria were shown to be able to activate AAI and AAII to reactive species, which were trapped with exogenous calf thymus DNA and analysed by postlabelling. Incubation of AAI and AAII in explanted rat stomach held in short-term organ culture resulted in DNA adduct formation in the epithelia of both forestomach and glandular stomach. To assign the recently characterized in vitro nucleoside adducts of AAI to the bisphosphate derivatives, a new ion-pair HPLC procedure on a reversed-phase column was developed. By monitoring Cerenkov radiation on-line, a good separation of AAI adducts was observed, demonstrating that adducts formed in vivo were chromatographically indistinguishable with those formed in vitro, and previously characterized as an aristolactam I moiety bound covalently to the exocyclic amino groups of deoxyadenosine and deoxyguanosine.

Aristolochic acid binds covalently to the exocyclic amino group of purine nucleotides in DNA.

The plant extract aristolochic acid (AA) has been used as a herbal drug in many cultures since antiquity. In 1982 AA was shown to be mutagenic and a strong carcinogen in Wistar rats. The crude mixture consists of five nitrophenanthrene carboxylic acid derivatives with aristolochic acid I [AA I; 8-methoxy-6-nitro-phenanthro-(3,4-d)-1,3-dioxolo-5-carboxyli c acid] being the major component. The isolated compound has been found to be mutagenic in the Ames assay. The major metabolite of AA I formed under anaerobic conditions in vitro and excreted in vivo in several species including man, is the reduction product aristolactam I. Using the 32P-postlabeling assay, we could show that AA I forms covalent DNA adducts upon metabolic activation in vitro and in vivo in different organs in the rat. Xanthine oxidase, a mammalian nitroreductase, has served as a sufficient model system mimicking the reductive route of in vivo activation of carcinogenic nitroarenes. This paper reports on two major fluorescent adducts of AA I formed by in vitro reaction of AA I with xanthine oxidase and deoxyguanosine or deoxyadenosine. After isolation and purification by preparative HPLC the adducts were characterized by 1H-NMR, FAB mass, UV/Vis and fluorescence spectroscopy. Their structures were elucidated as 7-(deoxyguanosin-N2-yl)-aristolactam I and 7-(deoxyadenosin-N6-yl)-aristolactam I. These findings are in marked contrast to the results reported for other nitroaromatic carcinogens, where C8-modified deoxyguanosine adducts predominate and N2-substituted deoxyguanosine derivatives are found as minor reaction products. Our results suggest a cyclic N-acylnitrenium ion with delocalized positive charge as the ultimate carcinogenic species, binding preferentially to the exocyclic amino group of purine nucleotides in DNA.