Repair of O6-carboxymethylguanine adducts by O6-methylguanine-DNA methyltransferase in human colon epithelial cells.

The protein O6-methylguanine-DNA methyltransferase (MGMT) is able to repair the mutagenic O6-methylguanine (O6-MeG) adduct back to guanine. In this context, it may protect against colorectal cancer formation associated with N-nitroso compounds. Such compounds may be endogenously formed by nitrosylation of amino acids, which can give rise to mutagenic O6-MeG and O6-carboxymethylguanine (O6-CMG) adducts. It is well established that O6-MeG is repaired by MGMT. However, up to now, whether O6-CMG is repaired by this enzyme remains unresolved. Therefore, the aim of the present study was to analyze the fate of both types of O6-guanine adducts in the presence and absence of MGMT activity. To this end, MGMT activity was efficiently blocked by its chemical inhibitor O6-benzylguanine in human colon epithelial cells (HCECs). Exposure of cells to azaserine (AZA) caused significantly higher levels of both O6-MeG and O6-CMG adducts in MGMT-inhibited cells, with O6-CMG as the more abundant DNA lesion. Interestingly, MGMT inhibition did not result in higher levels of AZA-induced DNA strand breaks in spite of elevated DNA adduct levels. In contrast, MGMT inhibition significantly increased DNA strand break formation after exposure to temozolomide (TMZ), a drug that exclusively generates O6-MeG adducts. In line with this finding, the viability of the cells was moderately reduced by TMZ upon MGMT inhibition, whereas no clear effect was observed in cells treated with AZA. In conclusion, our study clearly shows that O6-CMG is repaired by MGMT in HCEC, thereby suggesting that MGMT might play an important role as a tumor suppressor in diet-mediated colorectal cancer.

Non-targeted and targeted analysis of oxylipins in combination with charge-switch derivatization by ion mobility high-resolution mass spectrometry.

Eicosanoids and other oxylipins play an important role in mediating inflammation as well as other biological processes. For the investigation of their biological role(s), comprehensive analytical methods are necessary, which are able to provide reliable identification and quantification of these compounds in biological matrices. Using charge-switch derivatization with AMPP (N-(4-aminomethylphenyl)pyridinium chloride) in combination with liquid chromatography ion mobility quadrupole time-of-flight mass spectrometry (LC-IM-QTOF-MS), we developed a non-target approach to analyze oxylipins in plasma, serum, and cells. The developed workflow makes use of an ion mobility resolved fragmentation to pinpoint derivatized molecules based on the cleavage of AMPP, which yields two specific fragment ions. This allows a reliable identification of known and unknown eicosanoids and other oxylipins. We characterized the workflow using 52 different oxylipins and investigated their fragmentation patterns and ion mobilities. Limits of detection ranged between 0.2 and 10.0 nM (1.0-50 pg on column), which is comparable with other state-of-the-art methods using LC triple quadrupole (QqQ) MS. Moreover, we applied this strategy to analyze oxylipins in different biologically relevant matrices, as cultured cells, human plasma, and serum. Graphical abstract.

Synthesis and in vitro characterization of the genotoxic, mutagenic and cell-transforming potential of nitrosylated heme.

Data from epidemiological studies suggest that consumption of red and processed meat is a factor contributing to colorectal carcinogenesis. Red meat contains high amounts of heme, which in turn can be converted to its nitrosylated form, NO-heme, when adding nitrite-containing curing salt to meat. NO-heme might contribute to colorectal cancer formation by causing gene mutations and could thereby be responsible for the association of (processed) red meat consumption with intestinal cancer. Up to now, neither in vitro nor in vivo studies characterizing the mutagenic and cell transforming potential of NO-heme have been published due to the fact that the pure compound is not readily available. Therefore, in the present study, an already existing synthesis protocol was modified to yield, for the first time, purified NO-heme. Thereafter, newly synthesized NO-heme was chemically characterized and used in various in vitro approaches at dietary concentrations to determine whether it can lead to DNA damage and malignant cell transformation. While NO-heme led to a significant dose-dependent increase in the number of DNA strand breaks in the comet assay and was mutagenic in the HPRT assay, this compound tested negative in the Ames test and failed to induce malignant cell transformation in the BALB/c 3T3 cell transformation assay. Interestingly, the non-nitrosylated heme control showed similar effects, but was additionally able to induce malignant transformation in BALB/c 3T3 murine fibroblasts. Taken together, these results suggest that it is the heme molecule rather than the NO moiety which is involved in driving red meat-associated carcinogenesis.

The influence of chronic L-carnitine supplementation on the formation of preneoplastic and atherosclerotic lesions in the colon and aorta of male F344 rats.

L-Carnitine, a key component of fatty acid oxidation, is nowadays being extensively used as a nutritional supplement with allegedly "fat burning" and performance-enhancing properties, although to date there are no conclusive data supporting these claims. Furthermore, there is an inverse relationship between exogenous supplementation and bioavailability, i.e., fairly high oral doses are not fully absorbed and thus a significant amount of carnitine remains in the gut. Human and rat enterobacteria can degrade unabsorbed L-carnitine to trimethylamine or trimethylamine-N-oxide, which, under certain conditions, may be transformed to the known carcinogen N-nitrosodimethylamine. Recent findings indicate that trimethylamine-N-oxide might also be involved in the development of atherosclerotic lesions. We therefore investigated whether a 1-year administration of different L-carnitine concentrations (0, 1, 2 and 5 g/l) via drinking water leads to an increased incidence of preneoplastic lesions (so-called aberrant crypt foci) in the colon of Fischer 344 rats as well as to the appearance of atherosclerotic lesions in the aorta of these animals. No significant difference between the test groups regarding the formation of lesions in the colon and aorta of the rats was observed, suggesting that, under the given experimental conditions, L-carnitine up to a concentration of 5 g/l in the drinking water does not have adverse effects on the gastrointestinal and vascular system of Fischer 344 rats.

The Resveratrol Tetramer r-Viniferin Induces a Cell Cycle Arrest Followed by Apoptosis in the Prostate Cancer Cell Line LNCaP.

Polyphenols are secondary plant metabolites that possess potentially health-promoting properties and which occur in various edible plants and plant products. Especially the stilbenoid resveratrol has been extensively studied regarding its anticarcinogenic and chemopreventive activities. However, research has recently focused on the investigation of other natural or synthetic compounds in order to find substances that show a higher bioactivity and/or bioavailability than resveratrol. In this context, we exemplarily investigated the cytotoxic/growth-inhibiting properties of the resveratrol tetramer r-viniferin on the prostate cancer cell line LNCaP and compared them with those of resveratrol. By using the sulforhodamine B assay followed by cell cycle analysis via flow cytometry and commercially available apoptosis/necrosis assay kits, we show that both compounds were able to inhibit the growth of LNCaP cells and to induce a cell cycle arrest in the G1 phase. However, r-viniferin was significantly more potent in inhibiting cellular growth than resveratrol and the only compound that increased the apoptotic cellular fraction as well as the activity of apoptosis-associated enzymes. In conclusion, r-viniferin leads to cytotoxicity in LNCaP cells at fairly low concentrations, and it is therefore conceivable that it might be used as a chemopreventive agent or as an adjuvant in prostate cancer therapy.

Caco-2/HT29-MTX co-cultured cells as a model for studying physiological properties and toxin-induced effects on intestinal cells.

Infectious gastrointestinal diseases are frequently caused by toxins secreted by pathogens which may impair physiological functions of the intestines, for instance by cholera toxin or by heat-labile enterotoxin. To obtain a functional model of the human intestinal epithelium for studying toxin-induced disease mechanisms, differentiated enterocyte-like Caco-2 cells were co-cultured with goblet cell-like HT29-MTX cells. These co-cultures formed a functional epithelial barrier, as characterized by a high electrical resistance and the presence of physiological intestinal properties such as glucose transport and chloride secretion which could be demonstrated electrophysiologically and by measuring protein expression. When the tissues were exposed to cholera toxin or heat-labile enterotoxin in the Ussing chamber, cholera toxin incubation resulted in an increase in short-circuit currents, indicating an increase in apical chloride secretion. This is in line with typical cholera toxin-induced secretory diarrhea in humans, while heat-labile enterotoxin only showed an increase in short-circuit-current in Caco-2 cells. This study characterizes for the first time the simultaneous measurement of physiological properties on a functional and structural level combined with the epithelial responses to bacterial toxins. In conclusion, using this model, physiological responses of the intestine to bacterial toxins can be investigated and characterized. Therefore, this model can serve as an alternative to the use of laboratory animals for characterizing pathophysiological mechanisms of enterotoxins at the intestinal level.

Dietary Polyphenols Inhibit the Cytochrome P450 Monooxygenase Branch of the Arachidonic Acid Cascade with Remarkable Structure-Dependent Selectivity and Potency.

The products of the cytochrome P450 monooxygenase (CYP)-catalyzed oxidation of arachidonic acid (AA), that is, epoxy- and hydroxy-fatty acids, play a crucial role in the homeostasis of several physiological processes. In a liver microsome-based multienzyme assay using AA as natural substrate, we investigated how polyphenols inhibit different oxylipin-forming CYP in parallel but independently from each other. The ω-hydroxylating CYP4F2 and CYP4A11 were investigated, as well as the epoxidizing CYP2C-subfamily and CYP3A4 along with the (ω-n)-hydroxylating CYP1A1 and CYP2E1. The oxylipin formation was inhibited by several polyphenols with a remarkable selectivity and a potency comparable to known CYP inhibitors. The flavone apigenin inhibited the epoxidation, ω-hydroxylation, and (ω-n)-hydroxylation of AA with IC50 values of 4.4-9.8, 2.9-10, and 10-25 µM, respectively. Other flavones such as wogonin selectively inhibited CYP1A1-catalyzed (ω-n)-hydroxylation with an IC50 value of 0.10-0.22 µM, while the isoflavone genistein was a selective ω-hydroxylase inhibitor (IC50: 5.5-46 µM). Of note, the flavanone naringenin and the anthocyanidin perlargonidin did not inhibit CYPs of the AA cascade. Moderate permeability of apigenin as tested in the Caco-2 model of intestinal absorption (Papp: 4.5 ± 1 × 10-6 cm/s) and confirmation of the inhibition of 20-HETE formation by apigenin in the colorectal cancer-derived cell line HCT 116 (IC50: 1.5-8.8 µM) underline the possible in vivo relevance of these effects. Further research is needed to better understand how polyphenols impact human health by this newly described molecular mode of action.

Gut microbial transformation of the dietary mutagen MeIQx may reduce exposure levels without altering intestinal transport.

The mutagen and probable human carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) is metabolized in the colon to 9-hydroxyl-2,7-dimethyl-7,9,10,11-tetrahydropyrimido[2',1':2,3]imidazo[4,5-f]quinoxaline (MeIQx-M1) by conjugation with microbially generated acrolein. However, whether this microbiota-controlled process alters systemic exposure and hepatotoxicity of MeIQx remains unclear. The physiological relevance of this microbial transformation on the systemic exposure of MeIQx was investigated using an in vitro-in vivo extrapolation approach. To address whether microbial transformation influences intestinal transport of MeIQx, the intestinal uptake of MeIQx and its metabolite MeIQx-M1 was quantified using Ussing chambers mounted with different intestinal segments from male Fischer 344 rats. Up to 0.4% of both MeIQx and MeIQx-M1 were transported from the mucosal side to the serosal side of intestinal tissue within 90 min, suggesting that the intestinal uptake of both compounds is similar. With the uptake rates of both compounds, physiologically based pharmacokinetic (PBPK) modeling of the fate of MeIQx in the human body including microbial transformation of MeIQx was performed. Results indicate for the first time that high levels of microbe-derived acrolein would be required to significantly reduce systemic exposure of MeIQx in humans. Finally, neither MeIQx nor MeIQx-M1 were cytotoxic towards human liver HepaRG cells at dietary or higher concentrations of MeIQx. In summary, these findings suggest that gut microbial transformation of heterocyclic amines has the potential to influence systemic human exposure to some extent, but may require significant gut microbial production of acrolein and that further investigations are needed to understand physiological levels of acrolein and competing biotransformation pathways.

Effects of a Grapevine Shoot Extract Containing Resveratrol and Resveratrol Oligomers on Intestinal Adenoma Development in Mice: In Vitro and In Vivo Studies.

SCOPE: Evidence suggests that the dietary consumption of plant extracts containing polyphenols might help prevent the onset of cancers of the gastrointestinal tract. In the present study, the chemopreventive and antiproliferative efficacy of a grapevine shoot extract (Vineatrol®30) containing resveratrol and resveratrol oligomers is investigated in vivo and in vitro. METHODS AND RESULTS: The in vivo study is performed using ApcMin mice on a high-fat diet, which represents a model of human adenomatous polyposis, while the potential of the extract as well as some of its isolated constituents to inhibit intestinal adenoma cell proliferation in vitro is investigated using APC10.1 cells derived from an ApcMin mouse. Vineatrol®30 at a low (2.3 mg kg-1  diet) or high dose (476 mg kg-1  diet) reduces the adenoma number in male and adenoma volume in female animals. Furthermore, Vineatrol®30 as well as resveratrol and two resveratrol tetramers compromise the expansion of APC10.1 cells by reducing cell number, inducing cell cycle arrest, cellular senescence, and apoptosis. However, except for the extract, none of the isolated resveratrol oligomers is more efficacious than resveratrol in these cells. CONCLUSION: Vineatrol®30 may merit further investigation as a potential dietary gastrointestinal cancer chemopreventive agent in humans.

The influence of a chronic L-carnitine administration on the plasma metabolome of male Fischer 344 rats.

SCOPE: L-carnitine has been advertised as a fat-lowering and performance-enhancing supplement, although scientific evidence for its effectiveness is lacking. The uptake of about 1-2 g of L-carnitine per day may result in the formation of metabolites like trimethylamine-N-oxide (TMAO), which in turn may be converted to potential carcinogens or promote the development of cardiovascular diseases. METHODS AND RESULTS: To assess whether an L-carnitine supplementation changes overall metabolism or causes the formation of previously unknown metabolites, we analyzed plasma samples from Fischer 344 rats originating from a previous study using a multi-platform metabolomics approach comprising LC-MS/MS and GC×GC-MS methods. Despite an intake of up to 352 mg L-carnitine/kg body weight/day for 1 year, plasma concentrations of only 29 out of 359 metabolites were significantly influenced, the induced concentration changes being often comparatively small. Nevertheless, a clear dose-response relationship and a substantial concentration increase were observed for TMAO, i.e. a tenfold higher TMAO level was measured in the high-dose group when compared to the control (2.5 versus 25.0 µM). CONCLUSION: Although L-carnitine supplementation did not cause large changes in the plasma metabolome, a higher risk for cardiovascular disease due to chronically elevated TMAO plasma concentrations cannot be excluded.

Gut Microbial Transformation of the Dietary Imidazoquinoxaline Mutagen MelQx Reduces Its Cytotoxic and Mutagenic Potency.

The diverse community of microbes present in the human gut has emerged as an important factor for cancer risk, potentially by altering exposure to chemical carcinogens. In the present study, human gut bacteria were tested for their capacity to transform the carcinogenic heterocyclic amine 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MelQx). Eubacterium hallii, Lactobacillus reuteri, and Lactobacillus rossiae were able to convert MelQx to a new microbial metabolite characterized on the basis of high-resolution mass spectrometry and NMR as 9-hydroxyl-2,7-dimethyl-7,9,10,11-tetrahydropyrimido[2',1':2,3]imidazo[4,5-f]quinoxaline (MelQx-M1), resulting from conjugation with activated glycerol. Acrolein derived from the decomposition of 3-hydroxypropionaldehyde, which is the product of bacterial glycerol/diol dehydratase activity, was identified as the active compound responsible for the formation of MelQx-M1. A complex human gut microbial community obtained from invitro continuous intestinal fermentation was found to also transform MelQx to MelQx-M1. MelQx-M1 had slightly reduced cytotoxic potency toward human colon epithelial cells invitro, and diminished mutagenic potential toward bacteria after metabolic activation. As bacterially derived acrolein also transformed 2 other HCAs, namely 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 2-amino-3-methylimidazo[4,5-f]quinoline, these results generalize the capacity of gut microbiota to detoxify HCAs in the gut, potentially modulating cancer risk.

Methionine restriction inhibits chemically-induced malignant transformation in the BALB/c 3T3 cell transformation assay.

High consumption of red meat entails a higher risk of developing colorectal cancer. Methionine, which is more frequently a component of animal proteins, and folic acid are members of the one carbon cycle and as such important players in DNA methylation and cancer development. Therefore, dietary modifications involving altered methionine and folic acid content might inhibit colon cancer development. In the present study, the BALB/c 3T3 cell transformation assay was used to investigate whether methionine and folic acid are able to influence the malignant transformation of mouse fibroblasts after treatment with the known tumour initiator 3-methylcholanthrene. Three different methionine concentrations (representing a -40%, a "normal" and a +40% cell culture medium concentration, respectively) and two different folic acid concentrations (6 and 20 µM) were thereby investigated. Methionine restriction led to a decrease of type III foci, while enhancement of both methionine and folic acid did not significantly increase the cell transformation rate. Interestingly, the focus-lowering effect of methionine was only significant in conjunction with an elevated folic acid concentration. In summary, we conclude that the malignant transformation of mouse fibroblasts is influenced by methionine levels and that methionine restriction could be a possible approach to reduce cancer development.

HPLC Separation of Vitamin E and Its Oxidation Products and Effects of Oxidized Tocotrienols on the Viability of MCF-7 Breast Cancer Cells in Vitro.

Tocotrienols, a vitamin E subgroup, exert potent anticancer effects, but easily degrade due to oxidation. Eight vitamin E reference compounds, α-, ß-, γ-, or δ-tocopherols or -tocotrienols, were thermally oxidized in n-hexane. The corresponding predominantly dimeric oxidation products were separated from the parent compounds by diol-modified normal-phase HPLC-UV and characterized by mass spectroscopy. The composition of test compounds, that is, α-tocotrienol, γ-tocotrienol, or palm tocotrienol-rich fraction (TRF), before and after thermal oxidation was determined by HPLC-DAD, and MCF-7 cells were treated with both nonoxidized and oxidized test compounds for 72 h. Whereas all nonoxidized test compounds (0-100 µM) led to dose-dependent decreases in cell viability, equimolar oxidized α-tocotrienol had a weaker effect, and oxidized TRF had no such effect. However, the IC50 value of oxidized γ-tocotrienol was lower (85 µM) than that of nonoxidized γ-tocotrienol (134 µM), thereby suggesting that γ-tocotrienol oxidation products are able to reduce tumor cell viability in vitro.

Investigation of the absorption of resveratrol oligomers in the Caco-2 cellular model of intestinal absorption.

Resveratrol oligomers are biologically active polyphenols found in wine. No information about the bioavailability of these polyphenols is available. In order to discover if the resveratrol oligomers can pass the intestinal barrier, transport of the dimer ε-viniferin and the tetramer hopeaphenol was studied in the Caco-2 transwell system. A flux through the cell monolayer could neither be observed for ε-viniferin nor for hopeaphenol (apparent permeability coefficient (Papp)<1×10(-6)cms(-1)). In contrast, resveratrol showed a Papp of 11.9×10(-6)cms(-1). Nevertheless, about 16-30% of the oligomers were found in the lysed cellular fraction. This leads to the conclusion that the intestinal absorption rate of the two resveratrol oligomers, ε-viniferin and hopeaphenol, is low and negligible when compared to resveratrol. Therefore, it is unlikely that the oligomers could elicit a systemic biological effect after dietary intake. However, the compounds may act locally on the intestinal epithelium.

Development of a rapid LC-UV method for the investigation of chemical and metabolic stability of resveratrol oligomers.

Resveratrol, piceatannol, ε-viniferin, r-viniferin, r2-viniferin, and hopeaphenol are naturally occurring polyphenols, associated with potentially beneficial health effects. We developed a rapid liquid chromatography-ultraviolet detection (LC-UV) method, allowing for the simultaneous determination of these six compounds in biological samples in less than 2.5 min with standard LC equipment. Using this method for the assessment of the stability of the six analytes, we demonstrated that all stilbene polyphenols disappear rapidly in Dulbecco's modified Eagle's medium (e.g., half-life of resveratrol of 1 h). In contrast, the tetramer hopeaphenol was stable over the maximum incubation time of 72 h. In incubations with liver microsomes, ε-viniferin was rapidly glucuronidated, although to a lower extent than resveratrol. Hopeaphenol was not glucuronidated at all. Given that glucuronidation is the major metabolic pathway for polyphenols, hopeaphenol might exhibit significantly different pharmacokinetic properties than other polyphenols. When chemical and metabolic stability as well as biological activity of hopeaphenol are taken together, these findings warrant further investigation of this polyphenol.

Activity-guided isolation of resveratrol oligomers from a grapevine-shoot extract using countercurrent chromatography.

An activity-guided isolation of bioactive stilbenes has been carried out with the grapevine-shoot extract Vineatrol 30. After hexane precipitation of the polymeric constituents, the stilbene mixture was separated on a preparative scale using low-speed rotary countercurrent chromatography (LSRCCC). The antiproliferative activity of the separated LSRCCC fractions was then screened in the human cancer cell line A-431, and trans-resveratrol, trans-ε-viniferin, r-2-viniferin, hopeaphenol, and miyabenol C were identified as active principles. In addition, a new class of stilbene derivatives, which exhibit a γ-lactam ring structure and exert a weak growth-inhibiting activity in A-431 cells, has been identified.