Berry ellagitannins may not be sufficient for prevention of tumors in the rodent esophagus.

Biodirected fractionation is used to identify the active inhibitory constituents in berries for esophageal cancer in rats. The present study was undertaken to determine if ellagitannins contribute to the chemopreventive activity of an alcohol/water-insoluble (residue) fraction of berries. Rats consumed diets containing residue fractions of three berry types, that is, black raspberries (BRBs), strawberries (STRWs), and blueberries (BBs), that differ in their content of ellagitannins in the order BRB > STRW > BB. Animals were fed residue diets beginning 2 weeks before treatment with the esophageal carcinogen N-nitrosomethylbenzylamine (NMBA) and throughout the 30-week bioassay. Residue fractions from all three berry types were about equally effective in reducing NMBA tumorigenesis in the rat esophagus irrespective of their ellagitannin content (0.01-0.62 g/kg of diet). These results suggest that the ellagitannins may not be responsible for the chemopreventive effects of the alcohol/water-insoluble fraction of berries.

Anthocyanins in black raspberries prevent esophageal tumors in rats.

Diets containing freeze-dried black raspberries (BRB) suppress the development of N-nitrosomethylbenzylamine (NMBA)-induced tumors in the rat esophagus. Using bioassay-directed fractionation, the anthocyanins in BRB were found to be the most active constituents for down-regulation of carcinogen-induced nuclear factor-kappaB and activator protein-1 expression in mouse epidermal cells in vitro. The present study was undertaken, therefore, to determine if the anthocyanins contribute to the chemopreventive activity of BRB in vivo. F344 rats consumed diets containing either (a) 5% whole BRB powder, (b) an anthocyanin-rich fraction, (c) an organic solvent-soluble extract (a-c each contained approximately 3.8 micromol anthocyanins/g diet), (d) an organic-insoluble (residue) fraction (containing 0.02 mumol anthocyanins/g diet), (e) a hexane extract, and (f) a sugar fraction (e and f had only trace quantities of anthocyanins), all derived from BRB. Animals were fed diets 2 weeks before treatment with NMBA and throughout the bioassay. Control rats were treated with NMBA only. Animals were killed at week 30, and esophageal tumors were enumerated. The anthocyanin treatments (diet groups a-c) were about equally effective in reducing NMBA tumorigenesis in the esophagus, indicating that the anthocyanins in BRB have chemopreventive potential. The organic-insoluble (residue) fraction (d) was also effective, suggesting that components other than berry anthocyanins may be chemopreventive. The hexane and sugar diets were inactive. Diet groups a, b, and d all inhibited cell proliferation, inflammation, and angiogenesis and induced apoptosis in both preneoplastic and papillomatous esophageal tissues, suggesting similar mechanisms of action by the different berry components.

Indole-3-carbinol inhibits 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone plus benzo(a)pyrene-induced lung tumorigenesis in A/J mice and modulates carcinogen-induced alterations in protein levels.

We tested the chemopreventive efficacy of indole-3-carbinol (I3C), a constituent of Brassica vegetables, and its major condensation product, 3,3'-diindolylmethane (DIM), against lung tumorigenesis induced by a mixture of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and benzo[a]pyrene (BaP) in A/J mice. The mixture of NNK plus BaP (2 micromol each) was administered by gavage as eight weekly doses, whereas I3C (112 micromol/g diet) and DIM (2 and 30 micromol/g diet in experiments 1 and 2, respectively) were given in the diet for 23 weeks beginning at 50% of carcinogen treatment. I3C reduced NNK plus BaP-induced tumor multiplicity by 78% in experiment 1 and 86% in experiment 2; the respective reductions in tumor multiplicity by DIM were 5% and 66%. Using a quantitative proteomics method, isobaric tags for relative and absolute quantitation (iTRAQ) coupled with mass spectrometry, we identified and quantified at least 250 proteins in lung tissues. Of these proteins, nine showed differences in relative abundance in lung tissues of carcinogen-treated versus untreated mice: fatty acid synthase, transketolase, pulmonary surfactant-associated protein C (SP-C), L-plastin, annexin A1, and haptoglobin increased, whereas transferrin, alpha-1-antitrypsin, and apolipoprotein A-1 decreased. Supplementation of the diet of carcinogen-treated mice with I3C reduced the level of SP-C, L-plastin, annexin A1, and haptoglobin to that of untreated controls. These results were verified using immunoblotting. We show here that tumor-associated signature proteins are increased during NNK plus BaP-induced lung carcinogenesis, and I3C inhibits this effect, suggesting that the lung tumor chemopreventive activity of I3C might be related to modulation of carcinogen-induced alterations in protein levels.

Formation and accumulation of pyridyloxobutyl DNA adducts in F344 rats chronically treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and enantiomers of its metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 1) and its metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL, 2) are both potent pulmonary carcinogens in rats. The metabolism of NNK to NNAL is stereoselective and reversible, with (S)-NNAL being the major enantiomer formed from NNK. In rats, (R)-NNAL undergoes facile glucuronidation and is rapidly excreted in urine, whereas (S)-NNAL is preferentially retained in tissues and converted to NNK. We hypothesized that the lung carcinogenicity of NNK in the rat is due in part to the preferential retention of (S)-NNAL in the lung, the reconversion to NNK, and then the metabolic activation of NNK to pyridyloxobutyl (POB)-DNA adducts. We tested this hypothesis by treating male F344 rats with 10 ppm of NNK, (R)-NNAL, or (S)-NNAL in drinking water. After 1, 2, 5, 10, 16, or 20 weeks of treatment, POB-DNA adducts in liver and lung DNA were quantified by HPLC-ESI-MS/MS. At each time point, total adduct levels were higher in the lung than in the liver. O2-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O2-POB-dThd, 13) was the major adduct detected. Total adduct levels in the rats treated with (S)-NNAL were 0.6-1.3 times as great as those in the NNK group in the lung and 0.7-1.4 times in the liver, and 6-14 times higher than those in the (R)-NNAL group in the lung and 11-17 times in the liver. These results suggest that (S)-NNAL is stereoselectively retained in tissues. This study demonstrates for the first time the accumulation and persistence of specific POB-DNA adducts in the rat lung and liver during chronic treatment with NNK, (R)-NNAL, and (S)-NNAL and supports the hypothesis that the preferential retention of (S)-NNAL in the lung, followed by reconversion to NNK and then the metabolic activation of NNK is critical for lung carcinogenesis by NNK and NNAL.

Analysis of pyridyloxobutyl DNA adducts in F344 rats chronically treated with (R)- and (S)-N'-nitrosonornicotine.

NNN (1) is an esophageal carcinogen in rats. 2'-Hydroxylation of NNN is believed to be the major bioactivation pathway for NNN tumorigenicity. (S)-NNN is preferentially metabolized by 2'-hydroxylation in cultured rat esophagus, whereas there is no preference for 2'-hydroxylation versus 5'-hydroxylation in the metabolism of (R)-NNN. 2'-Hydroxylation of NNN generates the reactive intermediate 4-oxo-4-(3-pyridyl)butanediazohydroxide (8), resulting in the formation of pyridyloxobutyl (POB)-DNA adducts. On the basis of these observations, we hypothesized that (S)-NNN treatment would produce higher levels of POB-DNA adducts than that by (R)-NNN in the rat esophagus. We tested this hypothesis by treating male F344 rats with 10 ppm of (R)-NNN or (S)-NNN in drinking water. After 1, 2, 5, 10, 16, or 20 weeks of treatment, POB-DNA adducts in esophageal, liver, and lung DNA were quantified by HPLC-ESI-MS/MS. In the rat esophagus, (S)-NNN treatment generated levels of POB-DNA adducts 3-5 times higher than (R)-NNN treatment, which supports our hypothesis. 7-[4-(3-Pyridyl)-4-oxobut-1-yl]guanine (7-POB-Gua, 14) was the major adduct detected, followed by O2-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O2-POB-dThd, 11) and O2-[4-(3-pyridyl)-4-oxobut-1-yl]cytosine (POB-Cyt, 15). O6-[4-(3-Pyridyl)-4-oxobut-1-yl]-2'-deoxyguanosine (O6-POB-dGuo, 10) was not detected. The total POB-DNA adduct levels in the esophagus were 3-11 times higher than those in the liver for (R)-NNN and 2-6 times higher than those for (S)-NNN. In contrast to the esophagus and liver, (R)-NNN treatment produced more POB-DNA adducts than (S)-NNN treatment in the rat lung, which suggested an important role for cytochrome P450 2A3 in NNN metabolism in the rat lung. In both the liver and lung, O2-POB-dThd was the predominant adduct and accumulated during the experiment. The results of this study demonstrate that individual POB-DNA adducts form and persist in the esophagi, livers, and lungs of rats chronically treated with NNN enantiomers and demonstrate that (S)-NNN produces higher levels of POB-DNA adducts in the esophagus than (R)-NNN, suggesting that (S)-NNN is more tumorigenic than (R)-NNN to the rat esophagus.

Identification of an acetaldehyde adduct in human liver DNA and quantitation as N2-ethyldeoxyguanosine.

Acetaldehyde, an ubiquitous mutagen and carcinogen, could be involved in human cancer etiology. Because DNA adducts are important in carcinogenesis, we have used liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) to explore the presence in human liver DNA of the major acetaldehyde DNA adduct, N2-ethylidenedeoxyguanosine (1). DNA was isolated and enzymatically hydrolyzed in the presence of NaBH3CN, which quantitatively converts adduct 1 to N2-ethyldeoxyguanosine (N2-ethyl-dGuo, 2). [15N5]N2-Ethyl-dGuo was synthesized and used as an internal standard. Adduct 2 was enriched from the hydrolysate by solid phase extraction and analyzed by LC-ESI-MS/MS. Clear peaks were observed for adduct 2 in analyses of human liver DNA, calf thymus DNA, and rat liver DNA. These peaks were not observed, or were much smaller, when the NaBH3CN step was omitted. When the DNA was subjected to neutral thermal hydrolysis prior to NaBH3CN treatment, adduct 2 was not observed. Control experiments using [13C2]acetaldehyde demonstrated that adducts 1 and 2 were not formed as artifacts during DNA isolation and analysis. These results strongly indicate that adduct 1 is present in human liver DNA and demonstrate that it can be quantified as adduct 2. Levels of adduct 2 measured in 12 human liver samples were 534 +/- 245 fmol/micromol dGuo (mean +/- SD). The results of this study establish the presence of an acetaldehyde adduct in human liver DNA and suggest that it is a commonly occurring endogenous DNA adduct.

The gel-forming polysaccharide of psyllium husk (Plantago ovata Forsk).

The physiologically active, gel-forming fraction of the alkali-extractable polysaccharides of Plantago ovata Forsk seed husk (psyllium seed) and some derived partial hydrolysis products were studied by compositional and methylation analysis and NMR spectroscopy. Resolving the conflicting claims of previous investigators, the material was found to be a neutral arabinoxylan (arabinose 22.6%, xylose 74.6%, molar basis; only traces of other sugars). With about 35% of nonreducing terminal residues, the polysaccharide is highly branched. The data are compatible with a structure consisting of a densely substituted main chain of beta-(1-->4)-linked D-xylopyranosyl residues, some carrying single xylopyranosyl side chains at position 2, others bearing, at position 3, trisaccharide branches having the sequence L-Araf-alpha-(1-->3)-D-Xylp-beta-(1-->3)-l-Araf. The presence of this sequence is supported by methylation and NMR data, and by the isolation of the disaccharide 3-O-beta-D-xylopyranosyl-L-arabinose as a product of partial acid hydrolysis of the polysaccharide.

Identification of O2-substituted pyrimidine adducts formed in reactions of 4-(acetoxymethylnitrosamino)- 1-(3-pyridyl)-1-butanone and 4-(acetoxymethylnitros- amino)-1-(3-pyridyl)-1-butanol with DNA.

Metabolic hydroxylation of the methyl group of the tobacco specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) results in the formation of intermediates that can alkylate DNA. Similarly, metabolic hydroxylation of the 2'-position of the tobacco specific carcinogen N'-nitrosonornicotine gives DNA alkylating intermediates. The resulting pyridyloxobutyl and pyridylhydroxybutyl adducts with dGuo have been characterized, but there are no reports of pyrimidine adducts. Therefore, in this study, we investigated the reactions of 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKCH(2)OAc) and 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanol (NNALCH(2)OAc) with DNA, dCyd, and dThd. NNKCH(2)OAc and NNALCH(2)OAc are stable precursors to the products formed upon metabolic methyl hydroxylation of NNK and NNAL. Analysis by LC-ESI-SIM of enzyme hydrolysates of DNA that had been allowed to react with NNKCH(2)OAc and NNALCH(2)OAc demonstrated the presence of major adducts with dCyd and dThd. The dCyd adducts were thermally unstable, releasing 4-HPB (18) or 4-hydroxy-1-(3-pyridyl)-1-butanol (25) upon treatment at 100 degrees C, pH 7.0. The dThd adducts were stable under these conditions. The dCyd adduct of NNALCH(2)OAc was characterized by its MS and UV and by conversion upon neutral thermal hydrolysis to the corresponding Cyt adduct, which was identified by MS, UV, and NMR. The dCyd and Cyt adducts of NNKCH(2)OAc were similarly characterized. The dThd adduct of NNKCH(2)OAc was identified by MS, UV, and NMR. Treatment of this adduct with NaBH(4) gave material, which was identical to that produced upon reaction of NNALCH(2)OAc with DNA or dThd. These data demonstrate that the major pyrimidine adducts formed in the reactions of NNKCH(2)OAc with DNA are O(2)[4-(3-pyridyl)-4-oxobut-1-yl]dCyd (26) and O(2)[4-(3-pyridyl)-4-oxobut-1-yl]dThd (30) while those produced from NNALCH(2)OAc are O(2)[4-(3-pyridyl)-4-hydroxybut-1-yl]dCyd (28) andO(2)[4-(3-pyridyl)-4-hydroxybut-1-yl]dThd (31). Levels of these pyrimidine adducts of NNKCH(2)OAc in DNA were substantially greater than those of the dGuo adducts of NNKCH(2)OAc, based on MS peak area. Furthermore, 26 was identified as a major 4-HPB releasing adduct of NNKCH(2)OAc. These results suggest that pyrimidine adducts of tobacco specific nitrosamines may be important contributors to their mutagenic and carcinogenic activity.