Tissue distribution and metabolism of the putative cancer chemopreventive agent 3',4',5'-trimethoxyflavonol (TMFol) in mice.

3',4',5'-Trimethoxyflavonol (TMFol) is a synthetic flavonol with preclinical cancer chemopreventive properties. The hypothesis was tested that, in mice, p.o. administration of TMFol results in measureable levels of the parent in target tissues. A single oral dose (240 mg/kg) was administered to mice (n = 4 per time point) with time points ranging from 5 to 1440 min. TMFol and its metabolites were identified and quantitated in all tissues by high-performance liquid chromatography (HPLC). Plasma levels of TMFol were at the limit of quantification or below, although metabolites were identified. Peak levels of TMFol in the gastrointestinal tract and the prostate averaged 1671 ± 265 µg/g (5.3 µmol/g) and 6.0 ± 1.6 µg/g (18.4 nmol/g), and occurred 20 and 360 min post-dose, respectively. The area under the tissue concentration-time curve (AUC) for TMFol was greater than those of the metabolites, indicating that TMFol is relatively metabolically stable. Micromolar TMFol levels are easily achieved in the prostate and gastrointestinal tract, suggesting that TMFol might exert chemopreventive efficacy at these tissue sites. Further investigations are warranted to elucidate the potential chemopreventive potency of TMFol.

Evaluation of urinary ribonucleoside profiling for clinical biomarker discovery using constant neutral loss scanning liquid chromatography/tandem mass spectrometry.

The patterns and levels of urinary excreted ribonucleosides which reflect RNA turnover and metabolism in humans offer the potential for early detection of disease and monitoring of therapeutic intervention. A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method employing constant neutral loss (CNL) scanning for the loss of the ribose moiety (132 u) was used to detect ribonucleosides in human urine and to evaluate this analytical platform for biomarker research in clinical trials. Ribonucleosides were stable and not influenced by the time spent at room temperature prior to freezing or long-term storage at -80 °C. Matrix effects caused variation in the mass spectrometer response which was dependent on the concentration of the analysed urine sample. For the use of urinary ribonucleoside profiling in clinical biomarker studies, adjustment of the urine samples to a common concentration prior to sample preparation is therefore advocated. Changes in the mass spectrometer response should be accounted for by the use of an internal standard added after sample preparation. Diurnal variation exceeded inter-day variation of an individual's ribonucleoside profile, but inter-person differences were predominant and allowed the separation of individuals against each other in a multivariate space. Due to considerable diurnal variation the use of spot urine samples would introduce unnecessary variation and should be replaced by the collection of multiple spot urine samples across the day, where possible. Should such a protocol not be feasible, biological intra-day and inter-day variation must be considered and accounted for in the data interpretation.

Determination of anthocyanins in the urine of patients with colorectal liver metastases after administration of bilberry extract.

Anthocyanins possess cancer chemopreventive properties in preclinical models. Their clinical pharmacology is only poorly understood. In this pilot study, anthocyanins and their metabolites were analysed in the urine of two patients with colorectal liver metastases. They received a single dose of 1.88 g standardized bilberry extract (mirtoselect) via either nasogastric or nasojejunal tube intra-operatively during liver resection. HPLC-MS/MS and HPLC-UV analysis showed there were more anthocyanins and metabolites in the urine of the patient who received mirtoselect via the stomach than via the jejunum. This result is consistent with information obtained in rodents which suggests the stomach is the predominant site for anthocyanin absorption.

Determination of 3',4',5',5,7-pentamethoxyflavone in the plasma and intestinal mucosa of mice by HPLC with UV detection.

In a preliminary experiment 3',4',5',5,7-pentamethoxyflavone (PMF) inhibited adenoma development in Apc(Min) mice, a model of the human heritable condition familial adenomatous polyposis. An HPLC method for tricin was modified and validated to permit measurement of PMF in mouse plasma and intestinal mucosa. HPLC analysis was carried out on a Hypersil-BDS C(18) column with detection at 324 nm and tricin as internal standard. The assay was linear in the range of 100-2000 ng/mL plasma and 1.0-40 microg/mL mucosa. PMF in plasma was efficiently extracted using solid-phase columns. In the case of mucosa organic solvent protein precipitation displayed satisfactory accuracy and precision. The assay recovery at low, medium and high concentrations was between 85 and 103% for both biomatrices, with a relative standard deviation of <15%. The lower limits of quantitation for plasma and mucosa were 100 ng/mL and 1.0 microg/mL, respectively. This method allowed measurement of PMF steady-state median concentrations in plasma (1.08 nmol/mL, n = 11; 10th and 90th percentiles: 0.633 and 2.385 nmol/mL) and mucosa (108.5 nmol/g, n = 9; 10th and 90th percentiles: 38.9 and 164.4 nmol/g) in mice which had received PMF (0.2%, w/w) with their diet.

Evaluation of the cancer chemopreventive efficacy of silibinin in genetic mouse models of prostate and intestinal carcinogenesis: relationship with silibinin levels.

Silibinin, a flavonolignan from milk thistle seeds, possesses cancer chemopreventive properties in rodent models of carcinogenesis. We tested the hypotheses that silibinin or silipide, silibinin formulated with phospholipids, delays tumour development in TRAMP or Apc(Min) mice, genetic models of prostate or intestinal malignancies, respectively. Mice received silibinin or silipide with their diet (0.2% silibinin equivalents) from weaning. Intervention with silipide reduced the size of well differentiated TRAMP adenocarcinomas by 31%. Silipide and silibinin decreased the incidence of poorly differentiated carcinomas by 61% compared to mice on control diet. Silipide decreased plasma levels of insulin-like growth factor (IGF)-1 by 36%. Levels of circulating IGF binding protein (IGFBP)-3 in mice on silipide or silibinin were 3.9- or 5.9-fold, respectively, elevated over those in control TRAMP mice. In Apc(Min) mice silibinin, but not silipide, had only a marginal adenoma number-reducing effect. The results cautiously support the advancement of silipide to the stage of clinical investigation in prostate cancer.

Androgen manipulation alters oxidative DNA adduct levels in androgen-sensitive prostate cancer cells grown in vitro and in vivo.

Intracellular reactive oxygen species (ROS) may cause oxidative DNA damage, resulting in the formation of adducts such as 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) and the cyclic pyrimidopurinone N-1, N(2) malondialdehyde-2'-deoxyguanosine (M(1)dG). These adducts have been associated with carcinogenesis, genomic instability and clonal evolution. We tested two hypotheses in human prostate cancer cells grown in vitro and in a xenograft model: (1) treatment of androgen-sensitive cells with DHT increases levels of oxidative DNA adduct levels; (2) flutamide, a competitive androgen receptor antagonist, prevents DHT-induced changes. Levels of M(1)dG and 8-oxo-dG adducts were determined by immunoslot blot and liquid chromatography-tandem mass spectrometry. M(1)dG and 8-oxo-dG levels were significantly higher than control levels in LNCaP cells exposed to supra-physiological concentrations (25-100 nM) of DHT (both P<0.05 by ANOVA). Flutamide pre-treatment completely prevented this increase. In the xenograft model, tumour levels of M(1)dG were decreased by 46% (P=0.001 by Mann-Whitney Test) in flutamide-treated animals compared to controls. The changes demonstrated suggest that oxidative DNA adducts may serve as biomarkers of the efficacy of androgen manipulation in chemoprevention trials.

Consumption of silibinin, a flavonolignan from milk thistle, and mammary cancer development in the C3(1) SV40 T,t antigen transgenic multiple mammary adenocarcinoma (TAg) mouse.

Silibinin is a flavonolignan extracted from milk thistle with cancer chemopreventive activity in preclinical models of prostate and colorectal cancer. A milk thistle extract, of which silibin is a major component, has recently been shown to exacerbate mammary carcinogenesis in two rodent models. We tested the hypothesis that consumption of silibinin or silipide, a silibinin formulation with pharmaceutical properties superior to the unformulated agent, affect breast cancer development in the C3(1) SV40 T,t antigen transgenic multiple mammary adenocarcinoma mouse model. Mice received silibinin or silipide (0.2% silibinin equivalents) with their diet from weaning, and tumour development was monitored by weekly palpation and the number and weight of neoplasms at the end of the experiment. Intervention neither promoted, nor interfered with, tumour development. The result suggests that promotion of carcinogenesis is not a feature of silibinin consistent across rodent models of mammary carcinogenesis.

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.

Modulation of response to cancer chemotherapeutic agents by diet constituents: is the available evidence sufficiently robust for rational advice for patients?

BACKGROUND: Patients who are diagnosed with cancer want advice on how they may alter their diet or which diet supplements they should take to augment chemotherapy. METHODS: We conducted a review of the literature mostly from the last 15 years on the interaction between dietary constituents and antineoplastic therapy in preclinical rodent models and in clinical trials. RESULTS: Studies have explored the effect of predominantly antioxidant vitamins and folate on efficacy or toxicity mediated by cisplatin and anthracyclins. Cisplatin toxicity in rodents was ameliorated by vitamin E. The design of clinical studies of dietary agents in combination with cytotoxic agents has been very heterogeneous and results have been inconclusive. CONCLUSIONS: Whilst preclinical experiments hint at a potential benefit of certain dietary agents, the evidence emanating from clinical studies does not allow firm conclusions to be made. Future studies should explore physiological doses of dietary agent and include pharmacokinetic and pharmacodynamic measurements.

Phase I dose escalation pharmacokinetic study in healthy volunteers of resveratrol, a potential cancer chemopreventive agent.

The red grape constituent resveratrol possesses cancer chemopreventive properties in rodents. The hypothesis was tested that, in healthy humans, p.o. administration of resveratrol is safe and results in measurable plasma levels of resveratrol. A phase I study of oral resveratrol (single doses of 0.5, 1, 2.5, or 5 g) was conducted in 10 healthy volunteers per dose level. Resveratrol and its metabolites were identified in plasma and urine by high-performance liquid chromatography-tandem mass spectrometry and quantitated by high-performance liquid chromatography-UV. Consumption of resveratrol did not cause serious adverse events. Resveratrol and six metabolites were recovered from plasma and urine. Peak plasma levels of resveratrol at the highest dose were 539 +/- 384 ng/mL (2.4 micromol/L, mean +/- SD; n = 10), which occurred 1.5 h post-dose. Peak levels of two monoglucuronides and resveratrol-3-sulfate were 3- to 8-fold higher. The area under the plasma concentration curve (AUC) values for resveratrol-3-sulfate and resveratrol monoglucuronides were up to 23 times greater than those of resveratrol. Urinary excretion of resveratrol and its metabolites was rapid, with 77% of all urinary agent-derived species excreted within 4 h after the lowest dose. Cancer chemopreventive effects of resveratrol in cells in vitro require levels of at least 5 micromol/L. The results presented here intimate that consumption of high-dose resveratrol might be insufficient to elicit systemic levels commensurate with cancer chemopreventive efficacy. However, the high systemic levels of resveratrol conjugate metabolites suggest that their cancer chemopreventive properties warrant investigation.

Tissue distribution in mice and metabolism in murine and human liver of apigenin and tricin, flavones with putative cancer chemopreventive properties.

PURPOSE: The flavones apigenin and tricin, which occur in leafy vegetables and rice bran, respectively, possess cancer chemopreventive properties in preclinical rodent models. Their pharmacology is only poorly understood. We compared their tissue levels in mice in vivo and their metabolism in liver fractions in vitro. METHODS: Mice received apigenin or tricin (0.2%) with their diet for 5-7 days, and flavone levels were compared in the plasma, liver and gastrointestinal mucosa using HPLC-UV. Flavone metabolism was investigated in murine and human liver microsomes or cytosol in vitro co-incubated with uridine 5'-diphosphoglucuronic acid or 3'-phosphoadenosine-5' phosphosulfate. Flavone metabolites were characterized by on-line HPLC-mass spectrometry. RESULTS: After dietary administration of flavones for 7 days, levels of tricin in plasma, liver and mucosa exceeded those of apigenin by 350, 33 and 100%, respectively. Apigenin was more rapidly glucuronidated than tricin in liver microsomes, whilst tricin underwent swifter sulfonation than apigenin in liver cytosol. For either flavone the rate of glucuronidation was much faster than that of sulfonation. Flavone monoglucuronides and monosulfates were identified as metabolites in microsomal and cytosolic incubations, respectively. CONCLUSIONS: When consumed with the diet in mice tricin seems to be more available than apigenin in blood and tissues. Differences in their glucuronidation may account for their differential availability. Thus tricin may have a pharmacokinetic advantage over apigenin. This type of information may help decide which flavonoids to select for clinical development.

Comparison of systemic availability of curcumin with that of curcumin formulated with phosphatidylcholine.

PURPOSE: Curcumin, a major constituent of the spice turmeric, suppresses expression of the enzyme cyclooxygenase 2 (Cox-2) and has cancer chemopreventive properties in rodents. It possesses poor systemic availability. We explored whether formulation with phosphatidylcholine increases the oral bioavailability or affects the metabolite profile of curcumin. METHODS: Male Wistar rats received 340 mg/kg of either unformulated curcumin or curcumin formulated with phosphatidylcholine (Meriva) by oral gavage. Rats were killed at 15, 30, 60 and 120 min post administration. Plasma, intestinal mucosa and liver were analysed for the presence of curcumin and metabolites using HPLC with UV detection. Identity of curcumin and metabolites was verified by negative ion electrospray liquid chromatography/tandem mass spectrometry. RESULTS: Curcumin, the accompanying curcuminoids desmethoxycurcumin and bisdesmethoxycurcumin, and the metabolites tetrahydrocurcumin, hexahydrocurcumin, curcumin glucuronide and curcumin sulfate were identified in plasma, intestinal mucosa and liver of rats which had received Meriva. Peak plasma levels and area under the plasma concentration time curve (AUC) values for parent curcumin after administration of Meriva were fivefold higher than the equivalent values seen after unformulated curcumin. Similarly, liver levels of curcumin were higher after administration of Meriva as compared to unformulated curcumin. In contrast, curcumin concentrations in the gastrointestinal mucosa after ingestion of Meriva were somewhat lower than those observed after administration of unformulated curcumin. Similar observations were made for curcumin metabolites as for parent compound. CONCLUSION: The results suggest that curcumin formulated with phosphatidylcholine furnishes higher systemic levels of parent agent than unformulated curcumin.

Pilot study of oral silibinin, a putative chemopreventive agent, in colorectal cancer patients: silibinin levels in plasma, colorectum, and liver and their pharmacodynamic consequences.

Silibinin, a flavonolignan from milk thistle, has intestinal cancer chemopreventive efficacy in rodents. It is a strong antioxidant and modulates the insulin-like growth factor (IGF) system by increasing circulating levels of IGF-binding protein 3 (IGFBP-3) and decreasing levels of IGF-I. Here, the hypothesis was tested that administration of oral silibinin generates agent levels in human blood and colorectal and hepatic tissues consistent with pharmacologic activity. Patients with confirmed colorectal adenocarcinoma received silibinin formulated with phosphatidylcholine (silipide) at dosages of 360, 720, or 1,440 mg silibinin daily for 7 days. Blood and biopsy samples of normal and malignant colorectum or liver were obtained before dosing, and blood and colorectal or hepatic tissues were collected at resection surgery after the final silipide dose. Levels of silibinin were quantified by high-pressure liquid chromatography-UV, and plasma metabolites were identified by liquid chromatography-mass spectrometry. Blood levels of IGFBP-3, IGF-I, and the oxidative DNA damage pyrimidopurinone adduct of deoxyguanosine (M1dG) were determined. Repeated administration of silipide was safe and achieved levels of silibinin of 0.3 to 4 micromol/L in the plasma, 0.3 to 2.5 nmol/g tissue in the liver, and 20 to 141 nmol/g tissue in colorectal tissue. Silibinin monoglucuronide, silibinin diglucuronide, silibinin monosulfate, and silibinin glucuronide sulfate were identified in the plasma. Intervention with silipide did not affect circulating levels of IGFBP-3, IGF-I, or M1dG. The high silibinin levels achieved in the human colorectal mucosa after consumption of safe silibinin doses support its further exploration as a potential human colorectal cancer chemopreventive agent.

Breast cancer prevention by green tea catechins and black tea theaflavins in the C3(1) SV40 T,t antigen transgenic mouse model is accompanied by increased apoptosis and a decrease in oxidative DNA adducts.

Tea consumption is associated with a reduced risk of mammary cancer as reflected by epidemiological studies and experiments in carcinogen-induced rodent models of mammary carcinogenesis. We tested the hypothesis that green tea catechins (GTC) or theaflavins from black tea (BTT) interfere with mammary carcinogenesis in C3(1) SV40 T,t antigen transgenic multiple mammary adenocarcinoma (TAg) mice and that GTC/BTT affect tumor survival or oxidation status. TAg mice received GTC/BTT (0.05%) in drinking water for their lifetime. As compared to control mice, they survived longer and had smaller tumors. On microscopic inspection, the size of the largest tumor per mouse was decreased by 40-42% (p<0.01). GTC (0.01%) and BTT (0.05%) increased levels of cleaved caspase 3 in tumor tissue by 67 and 38%, respectively (p<0.05), intimating increased apoptosis. Tumor levels of the malondialdehyde-DNA adduct M1dG in mice receiving GTC or BTT (0.05%) were reduced by 78 (p<0.001) or 63% (p<0.05), respectively, as compared to controls. The results render the exploration of the breast cancer chemopreventive properties of tea preparations in humans worthwhile.

Quantitation of silibinin, a putative cancer chemopreventive agent derived from milk thistle (Silybum marianum), in human plasma by high-performance liquid chromatography and identification of possible metabolites.

Silibinin has recently received attention as a potential cancer chemopreventive agent because of its antiproliferative and anticarcinogenic effects. A simple and specific reversed-phase high-performance liquid chromatography method was developed and validated for the quantitation of silibinin in human plasma. Sample preparation involved simple protein precipitation, and separation was achieved on a Waters Atlantis C18 column with flow rate of 1.0 mL/min at 40 degrees C and UV detection at 290 nm. Silibinin was detected as two peaks corresponding to trans-diastereoisomers. The peak area was linear over the investigated concentration range (0-5000 ng/mL). The limits of detection were 2 and 1 ng/mL for the two diastereoisomers (d1 and d2), with a recovery of 53-58%. This method was utilized to detect silibinin in plasma of colorectal patients after 7 days of treatment with silipide (silibinin formulated with phosphatidyl choline).

Pharmacokinetics and pharmacodynamics of curcumin.

Curcuma spp. contain turmerin, essential oils, and curcuminoids, including curcumin. Curcumin [1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione] is regarded as the most biologically active constituent of the spice turmeric and it comprises 2-8% of most turmeric preparations. Preclinical data from animal models and phase I clinical studies performed with human volunteers and patients with cancer have demonstrated low systemic bioavailability following oral dosing. Efficient first-pass metabolism and some degree of intestinal metabolism, particularly glucuronidation and sulfation of curcumin, might explain its poor systemic availability when administered via the oral route. A daily oral dose of 3.6 g of curcumin is compatible with detectable levels of the parent compound in colorectal tissue from patients with cancer. The levels demonstrated might be sufficient to exert pharmacological activity. There appears to be negligible distribution of the parent drug to hepatic tissue or other tissues beyond the gastrointestinal tract. Curcumin possesses wide-ranging anti-inflammatory and anticancer properties. Many of these biological activities can be attributed to its potent antioxidant capacity at neutral and acidic pH, its inhibition of cell signaling pathways at multiple levels, its diverse effects on cellular enzymes, and its effects on cell adhesion and angiogenesis. In particular, curcumin's ability to alter gene transcription and induce apoptosis in preclinical models advocates its potential utility in cancer chemoprevention and chemotherapy. With regard to considerable public and scientific interest in the use of phytochemicals derived from dietary components to combat or prevent human diseases, curcumin is currently a leading agent.

PPARdelta status and mismatch repair mediated neoplasia in the mouse intestine.

BACKGROUND: Therapeutic regulation of PPARdelta activity using selective agonists has been proposed for various disorders. However, the consequences of altered peroxisome proliferator-activated receptor delta (PPARdelta) activity in the context of intestinal tumourigenesis remain somewhat unclear. Contradictory evidence suggesting PPARdelta either attenuates or potentiates intestinal neoplasia. To further investigate the PPARdelta dependency of intestinal tumourigenesis, we have analysed the consequences of PPARdelta deficiency upon intestinal neoplasia occurring in mice with impaired mismatch DNA repair. METHODS: Mice deficient for both PPARdelta and the mismatch repair gene Mlh1 were produced and the incidence and severity of intestinal neoplasia recorded. RESULTS: No significant differences between the control genotypes and the double mutant genotypes were recorded indicating that deficiency of PPARdelta does not modify impaired mismatch repair induced neoplasia. CONCLUSION: In contrast with the previously observed acceleration of intestinal neoplasia in the context of the ApcMin/+ mouse, PPARdelta deficiency does not alter the phenotype of mismatch repair deficiency. This data supports the notion that PPARdelta is not required for adenoma formation and indicate that any pro-tumourigenic effect of PPARdelta inactivation may be highly context dependent.

Differential modulation of cyclooxygenase-mediated prostaglandin production by the putative cancer chemopreventive flavonoids tricin, apigenin and quercetin.

OBJECTIVES: Diet-derived flavonoids possess cancer chemopreventive properties in preclinical models. The knowledge of the pharmacology of most flavonoids is insufficient to warrant their advancement to clinical evaluation. METHODS: Here the three flavonoids tricin from rice bran, apigenin from leafy vegetables, and quercetin from onions and apples, were compared in terms of their ability to modulate cyclooxygenase- (COX-) catalyzed prostaglandin E-2 (PGE-2) generation. Specifically their effects on the following parameters were studied: (1) COX enzyme activity, (2) COX-2 expression in human-derived colon cancer cells HCA-7, which express COX-2 constitutively, (3) phorbol ester-mediated COX-2 induction in human colon epithelial cells (HCEC), and (4) PGE-2 levels in cellular incubations. RESULTS: Tricin and quercetin inhibited enzyme activity in purified COX-1 and -2 preparations with IC50 values of near 1 (tricin) and 5 microM (quercetin). Apigenin at up to 25 microM did not affect COX enzyme activity. Flavonoids were incubated with cells for 6 or 24 h and COX-2 protein expression and PGE-2 levels were assessed by Western blot and competitive immunoassay, respectively. None of the agents affected constitutive COX-2 expression in HCA-7 cells. Apigenin, but not tricin or quercetin, down-regulated inducible COX-2 expression in HCEC cells on 6 h incubation. All three flavonoids reduced cellular levels of PGE-2 in the supernatant of HCA-7 cells at both time points and of HCEC cells at 6 h. CONCLUSIONS: The results demonstrate that these structurally similar flavonoids regulate COX-mediated PGE-2 production in different fashions. Their ability to attenuate prostanoid levels may contribute to their cancer chemopreventive efficacy.

Preliminary safety evaluation of the putative cancer chemopreventive agent tricin, a naturally occurring flavone.

PURPOSE: Naturally occurring flavonoids such as quercetin and genistein possess cancer chemopreventive properties in experimental models. However, adverse effects such as their mutagenicity confound their potential clinical usefulness. Furthermore in leukaemia cells some flavonoids cleave the breakpoint cluster region of the mixed lineage leukaemia (MLL) gene as a consequence of inhibition of topoisomerase II. The choice of flavonoids to be developed as cancer chemopreventive agents depends crucially on their safety. Here, we explored safety aspects of the novel flavone tricin, a constituent of rice bran and other grass species, which has recently been found to interfere with murine gastrointestinal carcinogenesis. METHODS: Evidence of pathological or morphological changes in liver, lung, heart, spleen, kidney, adrenal gland, pancreas or thymus tissues was studied in mice which received tricin, genistein or quercetin 1,000 mg/kg daily by the oral route on five consecutive days. The ability of tricin (50 microM) to cleave the MLL gene was studied in human leukaemia cells by Southern blotting, and its effect on human topoisomerase II activity was investigated in incubations with supercoiled DNA. The mutagenicity of tricin was assessed in the Salmonella/Escherichia coli assay, and its clastogenicity was adjudged by chromosomal aberrations in Chinese hamster ovary cells and occurrence of micronuclei in bone marrow erythrocytes in Swiss-Webster mice. RESULTS: Neither tricin, quercetin, or genistein caused pathological or morphological changes in any of the murine tissues studied. Tricin (50 microM) failed to cause MLL gene breakage, and it inhibited topoisomerase II only at 500 microM, but not at 10, 50 or 100 microM. Tricin lacked genotoxic properties in the systems studied here. CONCLUSION: The results tentatively suggest that tricin may be considered safe enough for clinical development as a cancer chemopreventive agent.

Development of analyses by high-performance liquid chromatography and liquid chromatography/tandem mass spectrometry of bilberry (Vaccinium myrtilus) anthocyanins in human plasma and urine.

Anthocyanins are potent antioxidants that may possess chronic disease preventive properties. Here, rapid, reliable, and reproducible solid-phase extraction, high-performance liquid chromatography (HPLC), and mass spectrometry techniques are described for the isolation, separation, and identification of anthocyanins in human plasma and urine. Recoveries of cyanidin-3-glucoside (C3G) were 91% from water, 71% from plasma, and 81% from urine. Intra- and interday variations for C3G extraction were 9 and 9.1% in plasma and 7.1 and 9.1% in urine and were less than 15% for all anthocyanins from a standardized bilberry extract (mirtoselect). Analysis of mirtoselect by HPLC with UV detection produced spectra with 15 peaks compatible with anthocyanin components found in mirtoselect within a total run time of 15 min. Chromatographic analysis of human urine obtained after an oral dose of mirtoselect yielded 19 anthocyanin peaks. Mass spectrometric analysis employing multiple reaction monitoring suggests the presence of unchanged anthocyanins and anthocyanidin glucuronide metabolites.