Animal models and analytical approaches for understanding the relationships between wine and cancer.

We used two approaches for studying the relationships between wine consumption, wine composition and cancer In the first approach, a transgenic mouse model of human neurofibromatosis, combined with the use of well-defined, chemically purified diets, showed that red wine contains nonalcoholic components that can delay tumor onset. In additional studies, catechin, the main monomeric polyphenol of red wine, delayed tumor onset in this mouse model in a positive, linear relationship when incorporated into the diet at levels of 0.5-4 mmol/kg diet. In the second approach, low doses of the chemical carcinogen 2-amino-1-methyl-6-phenylimidazo(4, 5-b)pyridine (PhlP) were administered to rats, and formation of DNA adducts was evaluated by accelerator mass spectrometry. Consumption of red wine solids (the residue from red wine remaining after removal of alcohol and water) and the wine polyphenol quercetin did not influence PhlP-DNA adduct levels or induce liver enzymes (glutathione-S-transferase and quinone reductase). However, quercetin did alter distribution of PhlP in the rat tissues compared to control animals and animals fed other potential dietary chemopreventive agents, including phenylethyl isothiocyanate and sulforaphane. These studies demonstrate the feasibility of these approaches for studying the chemopreventive potential of dietary components at physiologic levels in

Long-term kinetic study of beta-carotene, using accelerator mass spectrometry in an adult volunteer.

We present a sensitive tracer method, suitable for in vivo human research, that uses beta-[(14)C]carotene coupled with accelerator mass spectrometry (AMS) detection. Using this approach, the concentration-time course of a physiological (306 microgram 200 nCi) oral dose of beta-[(14)C]carotene was determined for 209 days in plasma. Analytes included beta-[(14)C]carotene, [(14)C]retinyl esters, [(14)C]retinol, and several [(14)C]retinoic acids. There was a 5.5-h lag between dosing and the appearance of (14)C in plasma. Labeled beta-carotene and [(14)C]retinyl esters rose and displayed several maxima with virtually identical kinetic profiles over the first 24-h period; elevated [(14)C]retinyl ester concentrations were sustained in the plasma compartment for >21 h postdosing. The appearance of [(14)C]retinol in plasma was also delayed 5.5 h postdosing and its concentration rose linearly for 28 h before declining. Cumulative urine and stool were collected for 17 and 10 days, respectively, and 57.4% of the dose was recovered in the stool within 48 h postdosing. The stool was the major excretion route for the absorbed dose. The turnover times (1/k(el)) for beta-carotene and retinol were 58 and 302 days, respectively. Area under the curve analysis of the plasma response curves suggested a molar vitamin A value of 0.53 for beta-carotene, with a minimum of 62% of the absorbed beta-carotene being cleaved to vitamin A.In summary, AMS is an excellent tool for defining the in vivo metabolic behavior of beta-carotene and related compounds at physiological concentrations. Further, our data suggest that retinyl esters derived from beta-carotene may undergo hepatic resecretion with VLDL in a process similar to that observed for beta-carotene.

Determination of blood folate using acid extraction and internally standardized gas chromatography-mass spectrometry detection.

Whole blood folate level is a superior indicator of folate nutritional status than serum/plasma level. Problems with and lack of confidence in results of current whole blood folate assays have limited its popularity for assessing folate nutritional status. Here, an acid extraction GCMS detection method that measures total folate whole blood is presented. Folates are released from the matrix of whole blood and cleaved to para-aminobenzoic acid (pABA) by acid hydrolysis in the presence of [(13)C(6)]pABA as internal standard (IS). The hydrolysate is passed over a C18 resin to remove heme. The pABA isotopomers are ethyl esterified, isolated on C18 resin, and trifluoroacetylated. Following normal-phase HPLC separation, the isotopomers are silylated to their tBDMS derivatives. The abundance of these derivatives are measured at m/z 324 for [(13)C(6)]pABA as IS and m/z 318 for pABA from whole blood folate. Our method uses readily available chemicals and our results agree well with those using Lactobacillus casei, the current gold standard reference assay. The presence of folate analogs (methotrexate) or antibacterials (sulfonamines) does not affect our method. This feature makes it useful in monitoring folate status of patients undergoing chemotherapy. Before using our method, pABA supplements must be discontinued for a few days.

Tissue folate binding protein levels in transgenic mice with tumors and in non-transgenic controls.

Localized folate deficiency may be a risk factor for cancer. Since, folate binding proteins (FBP) and reduced folate carrier proteins (RFC) mediate cellular transport of folate, we compared FBP concentrations in several organs from tumor-bearing transgenic (TBT) mice and tumor-free non-transgenic controls (NTC) of the same strain, age, and fed identical diets. Liver, spleen, brain, small intestine and kidney were individually homogenized in phosphate-buffered saline (PBS) and separated into membrane, cytoplasmic, mitochondrial/lysomal and nuclear fractions (confirmed with marker enzymes). Homogenates and fractions was analyzed for total protein, and FBP. We used rabbit anti-bovine milk antibody and ELISA to measure FBP. FBP concentrations in kidney, small intestine, and spleen of TBT mice were higher than those of NTC mice; the opposite was true in liver and lung. FBP seemed to be upregulated in kidneys (all fractions), small intestine (all fractions), and spleen (cytoplasmic and nuclear fractions only) of TBT mice compared to NTC mice; the opposite appeared true in liver (all fractions) and lung (all fractions). FBP concentrations in brain, heart, and muscle of TBT mice were not different from those in brain, heart and muscle of NTC mice. A longitudinal study will determine if these changes in FBP concentrations precede tumor onset.

Intrinsic erythrocyte labeling and attomole pharmacokinetic tracing of 14C-labeled folic acid with accelerator mass spectrometry.

Long-term physiologic tracing of nutrients, toxins, and drugs in healthy subjects is not possible using traditional decay counting of radioisotopes or stable isotope mass spectrometry due to radiation exposure and limited sensitivity, respectively. A physiologic dose of 14C-labeled folic acid (35 microg, 100 nCi) was ingested by a healthy adult male and followed for 202 days in plasma, erythrocytes, urine, and feces using accelerator mass spectrometry. All samples and generated wastes were classified nonradioactive and the subject received a lifetime-integrated radiological effective dose of only 11 microSv. Radiolabeled folate appeared in plasma 10 min after ingestion but did not appear in erythrocytes until 5 days later. Approximately 0.4% of the erythrocytes were intrinsically labeled with an average of 130 (14)C atoms during erythropoiesis from the pulse of plasma [14C]folate. An appropriate radiocarbon-labeled precursor can intrinsically label DNA or a specific protein during synthesis and obtain limits of quantitation several orders of magnitude below that of stable isotope methods.

The dynamics of folic acid metabolism in an adult given a small tracer dose of 14C-folic acid.

Folate is an essential nutrient that is involved in many metabolic pathways, including amino acid interconversions and nucleotide (DNA) synthesis. In genetically susceptible individuals and populations, dysfunction of folate metabolism is associated with severe illness. Despite the importance of folate, major gaps exist in our quantitative understanding of folate metabolism in humans. The gaps exist because folate metabolism is complex, a suitable animal model that mimics human folate metabolism has not been identified, and suitable experimental protocols for in vivo studies in humans are not developed. In general, previous studies of folate metabolism have used large doses of high specific activity tritium and 14C-labeled folates in clinical patients. While stable isotopes such as deuterium and 13C-labeled folate are viewed as ethical alternatives to radiolabeled folates for studying metabolism, the lack of sensitive mass spectrometry methods to quantify them has impeded advancement of the field using this approach. In this chapter, we describe a new approach that uses a major analytical breakthrough, Accelerator Mass Spectrometry (AMS). Because AMS can detect attomole concentrations of 14C, small radioactive dosages (nCi) can be safely administered to humans and traced over long periods of time. The needed dosages are sufficiently small that the total radiation exposure is only a fraction of the natural annual background radiation of Americans, and the generated laboratory waste may legally be classified non-radioactive in many cases. The availability of AMS has permitted the longest (202 d) and most detailed study to date of folate metabolism in a healthy adult human volunteer. Here we demonstrate the feasibility of our approach and illustrate its potential by determining empirical kinetic values of folate metabolism. Our data indicate that the mean sojourn time for folate is in the range of 93 to 120 d. It took > or = 350 d for the absorbed portion of small bolus dose of 14C-folic acid to be eliminated completely from the body.

Ion trap mass spectrometry for kinetic studies of stable isotope labeled vitamin A at low enrichments.

The role of beta-carotene in chemoprevention of cancers and other chronic diseases generated controversy when subpopulations taking beta-carotene supplements showed increased mortality in clinical trials. Determination of the dynamics of beta-carotene in individual human subjects has emerged as a high priority. Stable isotope labeled beta-carotene tracers can be employed to determine rates of conversion to retinol (vitamin A), but tracer doses must be small to minimize perturbation of endogenous retinoid and carotenoid pools. In such cases, ratios of labeled tracer/endogenous retinol are often low, and quantitative analysis at enrichments of < 1 mol% are unreliable owing to ion-molecule reactions that generate ions at the same mass as the labeled tracer even when no tracer is present. The current study demonstrates improved gas chromatography/mass spectrometry quantification of retinol-d4 and unlabeled retinol, as their tert-butyldimethylsilyl ethers, at low enrichments using an ion trap mass spectrometer operated in selected ion storage mode. Electron ionization of analyte takes place in the ion trap using conditions that eject ions outside the range m/z 390-420, and molecular ions at m/z 400 and 404 from retinol and retinol-d4 are quantified. Using this approach, unlabeled retinol yields a signal close to values calculated from natural isotopic abundances (approximately 0.13%), whereas several quadrupole instruments operated using selected ion monitoring yielded 2-5 times greater signal when no labeled retinol was present.

Quantitative analysis by gas chromatography of volatile carbonyl compounds in expired air from mice and human.

Formaldehyde, acetaldehyde and acetone expired from tumor-bearing transgenic mice and formaldehyde exhaled from breast cancer patients were analyzed using gas chromatography. The tumor-bearing mice expired significantly more formaldehyde per unit metabolic size (1.43-2.98 micromol) than did control mice (0.77-1.01 micromol). There was no detectable difference in the levels of expired acetaldehyde and acetone between the two groups of mice. The exhaled formaldehyde levels from three women with breast cancer and from three healthy women were satisfactorily determined using the method developed in this study. The results suggest that these carbonyl compounds may be used as a biomarker.

Delayed tumor onset in transgenic mice fed an amino acid-based diet supplemented with red wine solids.

Increased consumption of vegetable foods (cereals, legumes, fruits) and some beverages (tea, cider, wine) is associated with reduced risk of cancer. Polyphenols in these foods and beverages are thought to be responsible, based on data from in vitro assays and from in vivo studies that used animals pretreated with carcinogen and given tea or polyphenol-spiked water to drink. We tested the hypothesis that dehydrated-dealcoholized red wine (wine solids), when consumed as part of a precisely defined complete diet, would delay tumor onset in transgenic mice that spontaneously develop externally visible tumors without carcinogen pretreatment. Sibling transgenic mice were weaned onto an amino acid-based diet alone or supplemented with red wine solids. Mice were examined daily; the age at which a first tumor appeared was recorded as the age of tumor onset. The concentration of the major polyphenol of red wine (catechin) in blood serum was also measured at the end of the study. The supplemented diet was fed continuously for three generations to ensure that it supported normal growth and reproduction. We discovered that the wine solid supplement delayed tumor onset, that intact catechin was absorbed, and that the supplemented diet supported normal growth and reproduction for three generations. Also, our simple experimental protocol offers an alternate and/or complementary way to identify foods, beverages, and their constituents that delay tumor onset and to investigate possible mechanisms involved.

Behavioral and neurochemical changes in folate-deficient mice.

Weanling mice were fed an amino acid-based diet supplemented with 0 or 11.3 mumol folic acid/kg diet for approximately 38 days to study behavior and neurochemistry in folate deficiency. After approximately 5 wk, mice fed the unsupplemented diet weighted approximately 70% as much those fed the supplemented diet. After 2 wk, mice fed the unsupplemented diet consistently discarded (spilled) more food, and after approximately 5 wk, they had spilled 3 times more than mice fed the supplemented diet. Serum folate, brain folate and brain S-adenosylmethionine of mice fed the unsupplemented diet were 4, 53, and 60% as high, respectively, as those of mice fed the supplemented diet. Pathologic changes were not evident in brain, spinal cord, or skeletal muscle of folate-deficient mice. The hypothalamic 5-hydroxyindole acetic acid/serotonin ratio and caudate dopamine, homovanillic acid, and 3,4-dihydroxyphenylacetic acid concentrations were lower in deficient than control mice. Folate-deficient mice develop a behavioral activity, food spilling, which may have a neurochemical basis in the serotonin and dopamine systems.

Estimating derivatives of pharmacokinetic response curves with varying bandwidths.

A kernel-smoothing method with locally varying bandwidths for the nonparametric estimation of derivatives of a function is proposed for highly nonequidistant data as they occur in pharmacokinetic response curves. We construct estimates having the particular property that the derivative estimates correspond exactly to the corresponding derivatives of the curve estimate even under locally varying bandwidth choice. The effects on the estimation of peak location (characteristic points) are investigated. In an example, characteristic points are estimated for a recently developed in vivo isotope dilution assay for vitamin A (retinol) nutritional status. The in vivo kinetics of appearance and disappearance of isotopically labeled retinol can be described with the proposed method.

Volatile carbonyl levels in tissues of transgenic mice with nerve sheath tumors.

Volatile carbonyl compounds in homogenates prepared from various tissues of tumor-bearing transgenic mice were determined. Formaldehyde and acetaldehyde were derivatized to thiazolidines. Malonaldehyde was derivatized to 1-methylpyrazole. The derivatives were quantified by gas chromatography with a highly sensitive and specific nitrogen-phosphorus detector. The limits of quantitation of formaldehyde and malonaldehyde were 2 micrograms/ml of homogenate and 27 ng/ml of homogenate, respectively. Levels of malonaldehyde in the erythrocytes and gastrocnemius of tumor-bearing transgenic mice were elevated as compared to the same tissue in control non-transgenic mice. Brain, liver, kidney, heart, and spleen tissues of the tumor-bearing mice exhibited decreased malonaldehyde levels. Similar results were obtained for formaldehyde and acetaldehyde.

Effect of adenine metabolites on survival of Drosophila melanogaster of low xanthine dehydrogenase activity.

1. Low xanthine dehydrogenase (LXD) mutant Drosophila melanogaster were fed 0.2% adenine for 7 generations, no adenine for the next 2 generations (relaxed) and 0.2% adenine again for the next 3 generations (rechallenged) to obtain adenine-resistant lines of Drosophila (LXD-adenine). Flies grown without adenine served as LXD-controls. 2. Purines ranked as follows; adenine > adenosine > AMP > inosine > IMP in decreasing order of toxicity to LXD-adenine flies. 3. Addition of ribose to 9N position, or phosphate or carboxy to 6C position of the purine ring alleviated the toxicity. 4. More LXD-adenine offspring survived than did LXD-control offspring rechallenged with adenine.

Analysis of reactive carbonyls in the expired air of transgenic mice.

Methods for the determination of trace levels of volatile carbonyl compounds in air expired from mice were developed and validated. Tumor bearing transgenic mice or nontransgenic control mice were placed into a glass chamber through which air was passed continuously at 90 ml/min for 1 h. The effluent gas stream was bubbled into an aqueous cysteamine solution or an aqueous methylhydrazine solution. Formaldehyde, acetaldehyde, and acetone in expired air were derivatized to thiazolidine with cysteamine and malonaldehyde was derivatized to 1-methyl-2-pyrazole with methylhydrazine. The derivatized compounds were analyzed by capillary gas chromatography with flame photometric or nitrogen-phosphorous-specific detection. The lowest level quantitated was 4 micrograms/ml thiazolidine, equivalent to 1.35 micrograms/ml formaldehyde. Formaldehyde was recovered at a level of 1356 +/- 234 nmol/kg0.75 (mean +/- SD) from mice with tumors and 898 +/- 97 nmol/kg0.75 from mice without tumors, suggesting that tumor bearing transgenic mice expired significantly more formaldehyde than did tumor free controls. Amounts of expired acetaldehyde and acetone were not different among mice. Malonaldehyde was not detected in either group of mice.

Ineffective hematopoiesis in folate-deficient mice.

A folate-free amino acid-based diet provided an opportunity to characterize the effects of folate depletion on growth, tissue folate levels, and hematopoiesis of mice under well-standardized conditions. Weanling mice were fed a folate-free, amino acid-based diet supplemented with either 0 or 2 mg folic acid/kg diet for 35 to 48 days. Folate concentrations were decreased in liver, kidney, serum, and erythrocytes in mice fed the folate-free diet. The folate-deficient mice had anemia, reticulocytopenia, thrombocytopenia, and leukopenia, all of which reverted to normal after folic acid was reintroduced to the diet. Hematopoietic organs of folate-deficient mice had alterations that were similar to those seen in folate-deficient humans except that in mice, the hyperplasia of hematopoietic tissue occurred in the spleen rather than in the marrow. Ferrokinetic studies showed a normal 59Fe-transferrin half-life, but the percentage of 59Fe-incorporation into red blood cells at 48 hours was markedly subnormal. The number of committed hematopoietic progenitors at the stages of erythroid colony-forming units (CFUs), megakaryocyte CFUs, and granulocyte-macrophage CFUs were all increased in folate-deficient mice. However, the progeny of these progenitors was markedly decreased in folate-deficient mice. Thus, the folate-deficient mice had "ineffective hematopoiesis" leading to pancytopenia, and they therefore provide a murine model of megaloblastic anemia.

Folate deficiency alone does not produce neural tube defects in mice.

The incidence of neural tube defects was studied in mouse embryos from dams fed an amino acid-based diet containing 45, 91, 136, 181, 227 or 453 nmol folic acid/kg diet (Experiment 1) or 227, 453, 566, 680, 906, 1132, 1698 or 2266 nmol folic acid/kg diet (Experiment 2). Reproductive tracts were examined 12 d postcoitum and gross and microscopic examination of all embryos was performed. A single implantation was found at levels less than or equal to 181 nmol folic acid/kg diet. With one exception, bred mice fed 227 or 453 nmol folic acid/kg diet in Experiment 1 had 100% resorptions. In Experiment 2, 100% of implantations in mice fed 227 nmol folic acid/kg diet and approximately 75% of implantations in mice fed 453 or 566 nmol folic acid/kg diet resorbed. The 906 nmol folic acid/kg diet was sufficient for successful pregnancy. Mice fed 227 nmol folic acid/kg diet in Experiment 2 weighed approximately 80% of mice fed higher levels of folic acid. Inadequate dietary folic acid resulted in fewer and smaller embryos (which developed normally). These results suggest that folate deficiency alone is insufficient to produce neural tube defects in Swiss-Webster mice. Because individual micronutrients (e.g., folate) can be omitted from the amino acid-based diet, the specific role of folic acid in neurulation can now be studied systematically.

Delayed tumor onset in transgenic mice fed a low-folate diet.

BACKGROUND: Transgenic mice carrying the human T-lymphotropic virus type 1 tax1 (transactivator) gene develop peripheral nerve sheath tumors with well-characterized times of onset and tissue involvement. PURPOSE AND METHODS: To evaluate the effect of dietary folic acid on age at tumor onset and on the concentration of folate in tissues and tumors, we bred heterozygous transgenic mice and systematically assigned their offspring at weaning (within litters) to a 2 x 2 x 2 factorial arrangement. The three variables studied were 1) the tax1 gene (presence or absence), 2) gender (male or female), and 3) dietary level of folic acid (0.11 or 11.34 mumol folic acid per kilogram of controlled amino acid-based diet). Blood and tissues were collected from tumor-bearing transgenic mice (prior to cachexia) and from nontransgenic littermates, matched whenever possible for gender and diet. RESULTS: Transgenic mice fed a diet containing 0.11 mumol of folic acid per kilogram developed tumors significantly later (92.8 +/- 6.4 days) than did those fed a diet containing 11.34 mumol of folic acid per kilogram (71.9 +/- 3.9 days). Folate concentrations in tumors of mice fed the low-folate diet were approximately one third those in tumors of mice fed the higher folate diet. Brain folate concentrations in mice fed the low-folate diet were less than one half those in mice fed the higher folate diet. CONCLUSION: Results show that the onset of spontaneous tumors can be delayed by feeding mice the lowest level of folate adequate to meet nutritional requirements for normal growth. IMPLICATION: Transgenic animal models of human disease offer great potential for evaluating the role of micronutrients in human carcinogenesis.

Influence of folate status and polyphenol intake on thiamin status of Irish women.

The relationship of folate status and polyphenol intake to thiamin status was studied in 80 randomly selected elderly and young Irish women, with key variables affecting thiamin nutrition controlled for. Folate, thiamin, and polyphenol intakes were measured during a 4-wk baseline (elderly and young) and 6-wk double-blind (elderly) supplementation period. Only elderly subjects were randomly assigned to placebo, folic acid (400 micrograms), thiamin (10 mg), or folic-acid-plus-thiamin groups. Thiamin status (TPP effect) was not affected by folate status (plasma and erythrocyte folate) during the baseline period or with folic acid supplementation alone. Polyphenol intake was not correlated with thiamin status. Only thiamin intake and thiamin supplementation significantly affected thiamin status. Because the majority of subjects (102 of 160) were initially thiamin deficient, enrichment of Irish grain products with thiamin is recommended.