Dietary carbohydrates: a review of international recommendations and the methods used to derive them.

BACKGROUND/OBJECTIVES: Renewed dietary recommendations for carbohydrates have recently been published by various international health authorities. The present work (1) reviews the methods and processes (systematic approach/review, inclusion of public consultation) used to identify, select and grade the evidence underpinning the recommendations, particularly for total carbohydrate (CHO), fibre and sugar consumption, and (2) examines the extent to which variation in the methods and processes applied relates to any differences in the final recommendations. SUBJECTS/METHODS: A search of WHO, US, Canada, Australia and European sources identified 19 documents from 13 authorities with the desired detailed information. Processes and methods applied to derive recommendations were compiled and compared. RESULTS: (1) A relatively high total CHO and fibre intake and limited intake of (added or free) sugars are generally recommended. (2) Even where recommendations are similar, the specific justifications for quantitative/qualitative recommendations differ across authorities. (3) Differences in recommendations mainly arise from differences in the underlying definitions of CHO exposure and classifications, the degree to which specific CHO-providing foods and food components were considered, and the choice and number of health outcomes selected. (4) Differences in the selection of source material, time frames or data aggregation and grading methods appeared to have minor influence. CONCLUSIONS: Despite general consistency, apparent differences among the recommendations of different authorities would likely be minimized by: (1) More explicit quantitative justifications for numerical recommendations and communication of uncertainty, and (2) greater international harmonization, particularly in the underlying definitions of exposures and range of relevant nutrition-related outcomes.

Transcriptome analysis of peripheral blood mononuclear cells in human subjects following a 36 h fast provides evidence of effects on genes regulating inflammation, apoptosis and energy metabolism.

There is growing interest in the potential health benefits of diets that involve regular periods of fasting. While animal studies have provided compelling evidence that feeding patterns such as alternate-day fasting can increase longevity and reduce incidence of many chronic diseases, the evidence from human studies is much more limited and equivocal. Additionally, although several candidate processes have been proposed to contribute to the health benefits observed in animals, the precise molecular mechanisms responsible remain to be elucidated. The study described here examined the effects of an extended fast on gene transcript profiles in peripheral blood mononuclear cells from ten apparently healthy subjects, comparing transcript profiles after an overnight fast, sampled on four occasions at weekly intervals, with those observed on a single occasion after a further 24 h of fasting. Analysis of the overnight fasted data revealed marked inter-individual differences, some of which were associated with parameters such as gender and subject body mass. For example, a striking positive association between body mass index and the expression of genes regulated by type 1 interferon was observed. Relatively subtle changes were observed following the extended fast. Nonetheless, the pattern of changes was consistent with stimulation of fatty acid oxidation, alterations in cell cycling and apoptosis and decreased expression of key pro-inflammatory genes. Stimulation of fatty acid oxidation is an expected response, most likely in all tissues, to fasting. The other processes highlighted provide indications of potential mechanisms that could contribute to the putative beneficial effects of intermittent fasting in humans.

Can the rat be used as a valid model of human esophageal adenocarcinoma?

Animal models of Barrett's metaplasia and esophageal adenocarcinoma are important to further characterize the disease and test potential therapies. This paper reviews the development of the surgical model of esophageal adenocarcinoma in the rat and considers whether this model provides a biologically accurate representation of Barrett's esophagus and esophageal adenocarcinoma in humans.

Profiling CpG island field methylation in both morphologically normal and neoplastic human colonic mucosa.

Aberrant CpG island (CGI) methylation occurs early in colorectal neoplasia. Quantitative methylation-specific PCR profiling applied to biopsies was used to quantify low levels of CGI methylation of 18 genes in the morphologically normal colonic mucosa of neoplasia-free subjects, adenomatous polyp patients, cancer patients and their tumours. Multivariate statistical analyses distinguished tumour from mucosa with a sensitivity of 78.9% and a specificity of 100% (P=3 x 10(-7)). In morphologically normal mucosa, age-dependent CGI methylation was observed for APC, AXIN2, DKK1, HPP1, N33, p16, SFRP1, SFRP2 and SFRP4 genes, and significant differences in CGI methylation levels were detected between groups. Multinomial logistic regression models based on the CGI methylation profiles from normal mucosa correctly identified 78.9% of cancer patients and 87.9% of non-cancer (neoplasia-free+polyp) patients (P=4.93 x 10(-7)) using APC, HPP1, p16, SFRP4, WIF1 and ESR1 methylation as the most informative variables. Similarly, CGI methylation of SFRP4, SFRP5 and WIF1 correctly identified 61.5% of polyp patients and 78.9% of neoplasia-free subjects (P=0.0167). The apparently normal mucosal field of patients presenting with neoplasia has evidently undergone significant epigenetic modification. Methylation of the genes selected by the models may play a role in the earliest stages of the development of colorectal neoplasia.

The procyanidin-mediated induction of apoptosis and cell-cycle arrest in esophageal adenocarcinoma cells is not dependent on p21(Cip1/WAF1).

Previous studies have shown that the proanthocyanidin-mediated induction of apoptosis and arrest of the cell cycle in cancer cells was associated with up-regulation of p21(Cip1/WAF1) (p21), suggesting that p21 may be the molecular mediator of the observed effects. Here we show that procyanidins induce a rapid and sustained arrest of the cell cycle, and increase apoptosis, concomitant with an increase in p21 expression. However, blocking the PA-induced up-regulation of p21 expression with siRNA did not alter PA-mediated changes in apoptosis and cell cycle, demonstrating that p21 is not responsible for the PA-induced effects.

Development of a food frequency questionnaire for the assessment of quercetin and naringenin intake.

OBJECTIVES: To measure the relationship between quercetin and naringenin intakes as estimated by food frequency questionnaire (FFQ), and the urinary excretion of quercetin and naringenin aglycones after their enzymatic hydrolysis in human volunteers. SUBJECTS AND METHODS: Volunteers were recruited via the Human Nutrition Unit volunteer databank at the Institute of Food Research, Norwich. Sixty-three volunteers were recruited into the study, of which 14 were excluded and 49 completed the study. A modified FFQ was developed and used to estimate daily intake of quercetin and naringenin in 49 healthy volunteers who also provided five 24-h urine samples over a 2-week period. Urinary excretion of quercetin and naringenin metabolites was determined by solid-phase extraction and high-pressure liquid chromatography. RESULTS: The estimated mean intakes of quercetin and naringenin were 29.4 mg (s.d. 15.0) and 58.1 mg (s.d. 62.7) per day, respectively. Mean urinary excretion of quercetin was 60.1 microg (s.d. 33.1) and that of naringenin was 0.56 mg (s.d. 0.4). The correlation between FFQ estimated intake of quercetin and naringenin and levels excreted in the urine were r=0.82 (P<0.0001) and r=0.25 (P=0.05), respectively. CONCLUSIONS: We observed a statistically significant correlation between the urinary excretion of quercetin and naringenin metabolites and their dietary intake as estimated by FFQ. Use of FFQs in epidemiological studies requiring an estimate of flavonoid intake seems justified.

Review article: nutrition, obesity and colorectal cancer.

BACKGROUND: The age-adjusted incidence of colorectal cancer is higher in prosperous industrialized countries than elsewhere. Dietary factors may account for 75% of sporadic colorectal cancer in the west, but the mechanisms remain obscure. AIM: To review evidence for the effects of overweight and obesity, physical activity and specific dietary components on colorectal neoplasia. METHODS: English language papers cited on MEDLINE, obtained using search terms related to colorectal cancer, physical activity and body mass and specific food components were reviewed. RESULTS: There is evidence for adverse effects of overweight and obesity and protective effects of high physical activity against colon, but not for rectal cancer. These effects may reflect metabolic stress and chronic low-grade inflammation. There are also modest adverse effects of red and processed meat. There is evidence for protective effects of dietary fibre, but for fruits and vegetables the evidence remains weak and inconclusive. There is some evidence for protective effects of n-3 polyunsaturated fatty acids from fish, some micronutrients and possibly phytochemicals. The effects of many dietary constituents may depend upon genetic polymorphisms affecting a variety of genes. CONCLUSION: Further research should focus particularly on the effects of insulin-resistance, impaired glucose tolerance and chronic low-grade inflammation on the colonic mucosa.

Systematic review: Cyclo-oxygenase-2 in human oesophageal adenocarcinogenesis.

BACKGROUND: Published in vitro and animal in vivo studies have demonstrated that cyclo-oxygenase-2 plays an important role during oesophageal adenocarcinogenesis. However, the extent to which these studies are directly relevant to events in the human lower oesophagus is questionable. AIM: To perform a systematic review of all available human studies that have evaluated levels of cyclo-oxygenase-2 expression during the progression from Barrett's metaplasia to oesophageal adenocarcinoma. METHODS: A literature search was performed to identify all studies which qualitatively or quantitatively assessed cyclo-oxygenase-2 protein or gene expression in either Barrett's, dysplastic or adenocarcinoma tissue in humans. RESULTS: A total of 27 studies met the inclusion criteria. There was general agreement that cyclo-oxygenase-2 was either absent or very weakly expressed in normal oesophageal squamous mucosa, but considerable disagreement regarding the presence of cyclo-oxygenase-2 in Barrett's and low-grade dysplasia. All studies agreed that high-grade dysplasia and adenocarcinoma expressed cyclo-oxygenase-2 to some extent although levels varied considerably between tissue samples. CONCLUSIONS: There is conflicting evidence in the literature for cyclo-oxygenase-2 playing an important role in early oesophageal adenocarcinogenesis. Other non-cyclo-oxygenase-2 targets may account for the epidemiological data supporting the use of non-steroidal anti-inflammatory drugs in the chemoprevention of oesophageal adenocarcinoma.

Systematic review: the chemoprevention of oesophageal adenocarcinoma.

The incidence of oesophageal adenocarcinoma is increasing in the UK faster than any other malignancy. Despite its relatively poor prognosis and the limited success of existing treatments, there is enthusiasm that chemopreventive agents might be able to stem the transition from normal squamous epithelium to adenocarcinoma. We discuss gastro-oesophageal reflux as the main risk factor for the development of Barrett's metaplasia, the only known precursor of oesophageal adenocarcinoma. Treatment options for reflux disease are considered with regard to their effects on cancer risk. Recent advances in the molecular and cell biology of Barrett's are outlined, and potential targets for chemoprevention examined. Available treatments for reflux disease have not convincingly altered the likelihood of cancer development. Epidemiological and animal studies support the use of non-steroidal anti-inflammatory drugs as potential chemopreventive agents. Dietary agents, however, have a more favourable side-effect profile and may prove to be an attractive alternative, although more work is needed to fully explore this prospect.

Methylation of the ESR1 CpG island in the colorectal mucosa is an 'all or nothing' process in healthy human colon, and is accelerated by dietary folate supplementation in the mouse.

ESR1 is frequently silenced by CGI (CpG island) methylation, both in human colorectal tumours and, in an age-dependent manner, in healthy mucosa. It is not clear, however, whether methylation of individual cytosines occurs randomly within the epithelial genome, or preferentially within individual cells as an 'all-or-nothing' phenomenon. CGI methylation can be quantified in human DNA residues recovered from faecal samples. We used bisulphite genomic sequencing of human DNA from this source and from a colorectal cancer cell line (SW48) to show that the ESR1 CGI is methylated in an allele-specific manner. This provides support for the 'all or none' mechanism for methylation of this gene, and shows how age-dependent methylation of the ESR1 CGI leads rapidly to silencing of the gene within the cells, and hence the colonic crypt within which it occurs. Preliminary studies with a rodent model suggest the rate of age-dependent methylation of ESR1 is modifiable by dietary folate.

Using smart card technology to monitor the eating habits of children in a school cafeteria: 1. Developing and validating the methodology.

OBJECTIVE: The aim of the study was to test the feasibility of using smart card technology to track the eating behaviours of nearly a thousand children in a school cafeteria. METHODS: Within a large boys' school a smart card based system was developed that was capable of providing a full electronic audit of all the individual transactions that occurred within the cafeteria. This dataset was interfaced to an electronic version of the McCance and Widdowson composition of foods dataset. The accuracy of the smart card generated data and the influence of portion size and wastage were determined empirically during two 5-day trials. RESULTS: The smart card system created succeeded in generating precise data on the food choices made by hundreds of children over an indefinite time period. The data was expanded to include a full nutrient analysis of all the foods chosen. The accuracy of this information was only constrained by the limitations facing all food composition research, e.g. variations in recipes, portion sizes, cooking practices, etc. Although technically possible to introduce wastage correction factors into the software, thereby providing information upon foods consumed, this was not seen as universally practical. CONCLUSION: The study demonstrated the power of smart card technology for monitoring food/nutrient choice over limitless time in environments such as school cafeterias. The strengths, limitations and applications of such technology are discussed.

Using smart card technology to monitor the eating habits of children in a school cafeteria: 2. The nutrient contents of all meals chosen by a group of 8- to 11-year-old boys over 78 days.

OBJECTIVE: The aim of the study was to test the abilities of the newly created smart card system to track the nutrient contents of foods chosen over several months by individual diners in a school cafeteria. METHODS: From the food choice and composition of food data sets, an Access database was created encompassing 30 diners (aged 8-11 years), 78 days and eight nutrients. Data were available for a total of 1909 meals. RESULTS: Based upon population mean values the cohort were clearly choosing meals containing higher than the recommended maximum amounts for sugar and lower than the recommended minimum amounts of fibre, iron and vitamin A. Protein and vitamin C contents of meals chosen were well above minimum requirements. Over the 1909 meals, nutrient requirements were met 41% of the time. CONCLUSIONS: The system created was very effective at continually monitoring food choices of individual diners over limitless time. The data generated raised questions on the common practice of presenting nutrient intakes as population mean values calculated over a few days. The impact of heavily fortified foods on such studies in general is discussed.

Using smart card technology to monitor the eating habits of children in a school cafeteria: 3. The nutritional significance of beverage and dessert choices.

OBJECTIVE: The consumption patterns of beverages and desserts features highly in the current debate surrounding children's nutrition. The aim of this study was to continuously monitor the choice of beverages and desserts made by nearly 1000 children in a school cafeteria. METHODS: A newly developed smart card system was used to monitor the food choices of diners (7-16-year-old boys) in a school cafeteria over 89 days. A wide variety of beverages and desserts were on offer daily. RESULTS: Despite coming from an affluent, well-educated demographic group, the boys' choices of beverages and desserts mirrored those of children in general. Buns and cookies were over 10 times more popular than fresh fruits and yogurts. Sugary soft-drinks were over 20 times more popular than fresh fruit drinks and milk combined. Appropriate choices could, over a month, reduce intake of added sugar by over 800 g and fat by over 200 g. CONCLUSION: The smart card system was very effective at monitoring total product choices for nearly 1000 diners. In agreement with a recent national school meal survey, where choice is extensive, children show a preference for products high in fat and/or sugar. The consequences of these preferences are discussed.

New approaches to the role of diet in the prevention of cancers of the alimentary tract.

Cancers of the alimentary tract are, collectively, amongst the major causes of morbidity and deaths from cancer across the world today. Of the 10 million new cases of cancer diagnosed in 2000, about 2.3 million were cancers of the pharynx, oesophagus, stomach or colorectum. Nevertheless, epidemiological studies indicate that cancers of the digestive organs are also amongst the most susceptible to modification by dietary factors. International variations in incidence suggest that round three quarters of all sporadic colorectal cancers are attributable to diet. Even within the relatively uniform environment of the European Union, there are variations in the incidence of colorectal and oesophageal cancers of about two- and six-fold, respectively. Carcinomas of the alimentary tract arise from epithelial cells via distinct sequences of neoplastic change, which require a large fraction of an individual's lifespan. The best characterised of these is the adenoma-carcinoma sequence of colorectal carcinogenesis, in which progressive loss of differentiation and normal morphology in a growing lesion is associated with the acquisition of somatic mutations, and of aberrant methylation of CpG-islands, leading to gene silencing. These molecular events are accompanied by functional changes, including increased mitosis and evasion of apoptosis. There is little evidence that diet exerts its effects primarily through food-borne carcinogens that can be identified and eliminated from the food-chain. It is far more probable that the adverse effects of diet are caused largely by over-consumption of energy, coupled with inadequate intakes of protective substances, including micronutrients, dietary fibre and a variety of phytochemicals. The latter are biologically active secondary plant metabolites, many of which modify cell proliferation and induce apoptosis in vitro. There is growing evidence that such effects also occur in vivo, and that they can suppress the progress of neoplasia. Carcinomas of the oesophagus, stomach and colon all appear to be partially preventable by diets rich in fruits and vegetables. Plant foods contain a variety of components including micronutrients, polyunsaturated fatty acids, and secondary metabolites such as glucosinolates and flavonoids, many of which can inhibit cell proliferation and induce apoptosis, and which may well act synergistically when combined in the human diet. The future challenge is to fully characterise and evaluate these effects at the cellular and molecular level, so at to exploit their full potential as protective mechanisms for the population as a whole.

Synthetic and naturally occurring COX-2 inhibitors suppress proliferation in a human oesophageal adenocarcinoma cell line (OE33) by inducing apoptosis and cell cycle arrest.

Epidemiological studies suggest that the use of NSAIDs and/or a high intake of fruit and vegetables reduce the risk of oesophageal adenocarcinoma. Since COX-2 is up-regulated in Barrett's oesophageal carcinogenesis, the protective effect of NSAIDs and natural food components might reflect COX-2 inhibition. We explored the effects of quercetin, a natural flavonoid with a potent COX-2 inhibitory activity, and two commercially available selective COX-2 inhibitors (NS-398 and nimesulide) on cell proliferation, apoptosis, PGE2 production and COX-2 mRNA expression in a human oesophageal adenocarcinoma cell line (OE33). Changes in the relative numbers of adherent and floating cells were quantified and apoptotic cells were identified using ethidium bromide and acridine orange staining under fluorescence microscopy. Flow cytometric analysis of adherent and floating cells was used to quantify apoptosis and to examine the effects of the agents on the cell cycle. After 48 h exposure at concentrations of > or =1 microM both COX-2 inhibitors and quercetin suppressed cell proliferation (P < 0.01) and increased the fraction of floating apoptotic cells. At higher concentrations (50 microM) and longer exposure (48 h) the effects of quercetin were significantly greater than those of the selective COX-2 inhibitors (P < 0.01). Cell cycle analyses showed that quercetin blocked cells in S phase, while the selective COX-2 inhibitors blocked cells in G1/S interphase. COX-2 mRNA expression was suppressed by quercetin and the synthetic COX-2 inhibitors in a time- and dose-dependent manner. Quercetin and the synthetic COX-2 inhibitors (10 microM) suppressed PGE2 production by approximately 70% after 24 h exposure (P < 0.001). We conclude that OE33 is a useful model for the study of COX-2 expression and associated phenomena in human adenocarcinoma cells. Synthetic COX-2 inhibitors and the food-borne flavonoid quercetin suppress proliferation, induce apoptosis and cell cycle block in human oesophageal adenocarcinoma cells in vitro, and future studies should assess their effects in vivo.

Functional evidence of a role for two-pore domain potassium channels in rat mesenteric and pulmonary arteries.

1. Experiments were performed to elucidate the mechanism by which alterations of extracellular pH (pH(o)) change membrane potential (E(M)) in rat mesenteric and pulmonary arteries. 2. Changing pH(o) from 7.4 to 6.4 or 8.4 produced a depolarisation or hyperpolarisation, respectively, in mesenteric and pulmonary arteries. Anandamide (10 microm) or bupivacaine (100 microm) reversed the hyperpolarisation associated with alkaline pH(o), shifting the E(M) of both vessels to levels comparable to that at pH 6.4. In pulmonary arteries, clofilium (100 microm) caused a significant reversal of hyperpolarisation seen at pH 8.4 but was without effect at pH 7.4. 3. K(+) channel blockade by 4-aminopyridine (4-AP) (5 mm), tetraethylammonium (TEA) (10 mm), Ba(2+) (30 microm) and glibenclamide (10 microm) depolarised the pulmonary artery. However, shifts in E(M) with changes in pH(o) remained and were sensitive to anandamide (10 microm), bupivacaine (100 microm) or Zn(2+) (200 microm). 4. Anandamide (0.3-60 microm) or bupivacaine (0.3-300 microm) caused a concentration-dependent increase in basal tone in pulmonary arteries. 5. RT-PCR demonstrated the expression of TASK-1, TASK-2, THIK-1, TRAAK, TREK-1, TWIK-1 and TWIK-2 in mesenteric arteries and TASK-1, TASK-2, THIK-1, TREK-2 and TWIK-2 in pulmonary arteries. TASK-1, TASK-2, TREK-1 and TWIK-2 protein was demonstrated in both arteries by immunostaining. 6. These experiments provide evidence for the presence of two-pore domain K(+) channels in rat mesenteric and pulmonary arteries. Collectively, they strongly suggest that modulation of TASK-1 channels is most likely to have mediated the pH-induced changes in membrane potential observed in these vessels, and that blockade of these channels by anandamide or bupivacaine generates a small increase in pulmonary artery tone.

Micronutrients and cancer.

Adverse dietary factors account for approximately 30% of all cancers. Overconsumption of energy is undoubtedly one of the major risk factors, but dietary composition is also very important. In particular, a low consumption of fruits and vegetables appears to double the risk of carcinomas of the lung and alimentary tract. Epidemiological studies suggest that high plasma levels of Se, carotenoids and ascorbic acid are protective against cancer. However, intervention studies with antioxidant nutrients have given mixed results, and it has not been established that the benefits of a high intake of fruits and vegetables are invariably related to the prevention of oxidative DNA damage. Folic acid supplementation appears to protect against colo-rectal neoplasia, probably by preventing mutations associated with the repair of uracil mis-incorporation. However, there are indications from animal studies that exposure to high levels of folic acid at certain stages of development may lead to epigenetic effects that are, as yet, poorly understood. There seems little doubt that micronutrients contribute to the protective effects of plant foods against cancers of the lung and alimentary tract, but it has not been established that these benefits can be achieved using supplements.

Hydrolysis by lactase phlorizin hydrolase is the first step in the uptake of daidzein glucosides by rat small intestine in vitro.

1. Isoflavones are naturally occurring oestrogenic compounds found in plants, where they exist in the glycosylated form. A proportion of ingested glycosides appears to be absorbed in the upper gastrointestinal tract, where enterocytes play an important role in their metabolism. 2. One hypothesis is that ingestion may involve hydrolysis by the luminally exposed enzyme lactase phlorizin hydrolase (LPH), an enzyme expressed specifically at the small intestinal brush border. 3. Using an everted sac preparation of rat jejunum and an inhibitor of LPH, we investigated the absorption of daidzein-O(7)-glucoside (daidzin) and the effect of LPH inhibition on this process. It was demonstrated that LPH plays a major role in the deglycosylation of daidzin. 4. The hydrolysis product, daidzein, is absorbed by epithelial cells and glucuronidated to daidzein-O(7)-glucuronide, which is subsequently exported primarily to the serosal (vascular) side of the tissue rather than to the luminal side. 5. A small but significant proportion of the intact glycoside is also transferred to the serosal compartment, and in the presence of an LPH inhibitor this was enhanced with a corresponding reduction in deglucosylation and glucuronidation. 6. The results indicate that that LPH plays an important role in the metabolism of glycosylated phytochemicals, and that the expression and activity of this enzyme in the small intestine can modify the profile of metabolites appearing in the circulation.

Cyclo-oxygenase-2 expression in Barrett's oesophageal carcinogenesis: an immunohistochemical study.

BACKGROUND: The incidence of Barrett's oesophageal adenocarcinoma is increasing more rapidly than any other malignancy in industrialized countries. Cyclo-oxygenase-2 appears to play an important role in gastrointestinal carcinogenesis. Previous studies on cyclo-oxygenase-2 expression in Barrett's oesophageal carcinogenesis have utilized tissue samples obtained from different patients. We sought a definitive comparison of cyclo-oxygenase-2 expression in the sequence of Barrett's metaplasia-dysplasia-adenocarcinoma within the same patients. METHODS: Paraffin-embedded oesophago-gastrectomy specimens from 20 patients, containing successive stages of Barrett's metaplasia, high-grade dysplasia and adenocarcinoma, were analysed for cyclo-oxygenase-2 expression by immunohistochemistry. RESULTS: Cyclo-oxygenase-2 was constitutively expressed in the basal layers of cells in the adjacent normal squamous oesophageal epithelium, but a higher cyclo-oxygenase-2 expression was observed in Barrett's metaplasia. A further increase in cyclo-oxygenase-2 expression was detected in high-grade dysplasia, but cyclo-oxygenase-2 was decreased in adenocarcinoma tissue, regardless of its stage or level of differentiation. CONCLUSIONS: Cyclo-oxygenase-2 expression is progressively increased when squamous oesophageal epithelium develops into Barrett's metaplastic epithelium and then into high-grade dysplasia, but appears to decrease when adenocarcinoma develops. These findings may be significant for an effective chemo-prevention strategy with selective cyclo-oxygenase-2 inhibitors.