Bernadene A. Magnuson

Anthocyanin-rich extracts inhibit multiple biomarkers of colon cancer in rats.

Abstract: The aim of the present study was to investigate the chemoprotective activity of anthocyanin-rich extracts (AREs) from bilberry (Vaccinium myrtillus L.), chokeberry (Aronia meloncarpa E.), and grape (Vitis vinifera) by assessing multiple biomarkers of colon cancer in male rats treated with a colon carcinogen, azoxymethane. Fischer 344 male rats were fed the AIN-93 diet (control) or AIN-93 diet supplemented with AREs for 14 wk. Biomarkers that were evaluated included the number and multiplicity of colonic aberrant crypt foci (ACF), colonic cell proliferation, urinary levels of oxidative DNA damage, and expression of cyclooxygenase (COX) genes. To assess the bioavailability, levels of anthocyanins in serum, urine, and feces were evaluated. Total ACF were reduced (P < 0.05) in bilberry, chokeberry, and grape diet groups compared with the control group. The number of large ACF was also reduced (P < 0.05) in bilberry and chokeberry ARE-fed rats. Colonic cellular proliferation was decreased in rats fed bilberry ARE and chokeberry ARE diets. Rats fed bilberry and grape ARE diets had lower COX-2 mRNA expression of gene. High levels of fecal anthocyanins and increased fecal mass and fecal moisture occurred in ARE-fed rats. There was also a significant reduction (P < 0.05) in fecal bile acids in ARE-fed rats. The levels of urinary 8-hydroxyguanosine were similar among rats fed different diets. These results support our previous in vitro studies suggesting a protective role of AREs in colon carcinogenesis and indicate multiple mechanisms of action.

Anthocyanin-rich extract from Aronia meloncarpa E induces a cell cycle block in colon cancer but not normal colonic cells.

Anthocyanin-rich extracts, potent antioxidants and commercially available food coloring agents, have been reported to inhibit growth of various cancer cell lines. We investigated the effect of semipurified anthocyanin-rich extract from fruits of Aronia meloncarpa, on normal colon and colon cancer cell lines. A 24-h exposure to 50 µg monomeric anthocyanin/ml of Aronia extract resulted in 60% growth inhibition of human HT-29 colon cancer cells. The treated cells showed a blockage at G1/G0 and G2/M phases of the cell cycle. The cell cycle arrest coincided with an increased expression of the p21WAF1 and p27KIP1 genes and decreased expression of cyclin A and B genes. Prolonged exposure to the extract resulted in no further change in the cell number, indicating a cytostatic inhibition of cell growth. NCM460 normal colon cells demonstrated <10% growth inhibition at the highest concentration of 50 µg/ml extract. A 35% decrease in the cyclooxygenase-2 gene expression was observed within 24 h of exposure of HT-29 cells but did not translate into decreased protein levels or protein activity. These results support the need for further research to identify the specific component(s) in this extract that suppress cancer cell growth and the genes affected by these natural compounds.

Acute, subchronic and genotoxicity studies conducted with Oligonol, an oligomerized polyphenol formulated from lychee and green tea extracts

Oligonol is a phenolic product derived from lychee fruit extract and green tea extract, containing catechin-type monomers and oligomers of proanthocyanidins, produced by a manufacturing process which converts polyphenol polymers into oligomers. The safety of Oligonol was assessed in acute and subchronic studies and genotoxicity assays. In a single dose acute study of Oligonol, male and female rats were administered 2000mg/kg body weight (bw) Oligonol in water by gavage. Oligonol caused no adverse effects and body weight gain and food consumption were within normal range, thus the LD(50) of Oligonol was determined to be greater than 2000mg/kg. A 90 day subchronic study (100, 300 and 1000mg/kgbw/day, oral gavage) in male and female rats reported no significant adverse effects in food consumption, body weight, mortality, clinical chemistry, haematology, gross pathology and histopathology. Similarly, no adverse effects were observed in mice fed diets providing 2, 20 or 200mg/kgbw Oligonol or 200mg/kgbw lychee polyphenol for 90 days. Oligonol did not show any potential to induce gene mutations in reverse mutation tests using Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2uvrA strains. Oligonol did not induce chromosomal aberrations in cultured Chinese hamster lung cells, but it showed increased polyploidy. In a micronucleus assay in mice, Oligonol did not induce any micronuclei or suppress bone marrow, indicating it does not cause chromosome aberrations. The results from these safety studies and previous reports support the safety of Oligonol for human consumption.

Intact Anthocyanins and Metabolites in Rat Urine and Plasma After 3 Months of Anthocyanin Supplementation

Abstract: Anthocyanins are polyphenols responsible for most red to purple colors in plants. Human consumption of these pigments is increasing because of their potential health benefits and use as natural colorants. With more than 600 different anthocyanins found in nature, the impact of chemical structure on their absorption and metabolism needs to be investigated. Urine and plasma samples were collected from 32 rats receiving control diet or chokeberry-, bilberry-, and grape-enriched (3.85 g cyanidin 3-galatoside equivalent/kg) diet for 14 wk. Below 2 μmol/l of anthocyanins and relatively higher levels of presumable metabolites were detected by high-performance liquid chromatography-photodiode array in the plasma. In the urine the total concentration of intact anthocyanins and methylated derivatives ranged from 17.4 (bilberry) to 52.6 (chokeberry) nmol/l. The type and number of anthocyanin glycosylations affected the absorption remarkably. Detection of an acylated anthocyanin in plasma and urine suggests bioavailability of these anthocyanin derivatives that are commonly found in commercially available colorants.

Inhibition of Colonic Aberrant Crypt Foci by Curcumin in Rats Is Affected by Age

Curcumin has antioxidative, anti-inflammatory, and chemopreventive activities. To determine whether aging affects the inhibition of colon carcinogenesis by curcumin, young (6 wk), mature (12 mo), and old (22 mo) F344 male rats were fed either AIN-93 containing 0.6% curcumin or AIN-93 control diet. Aberrant crypt foci (ACF) were induced with two weekly s.c. injections of azoxymethane. After an additional 3 mo on the diets, the number, multiplicity, and distribution of ACF were evaluated. Addition of curcumin to the diet reduced the number of ACF by 49% in young rats and by 55% in old rats (P < 0.05). However, interestingly, no reduction of ACF was found in mature rats fed curcumin. Inhibition of large ACF was also affected by age, with the greatest reduction of large ACF occurring in old rats. However, animal age did not significantly alter the effect of dietary curcumin on reduction of cyclooxygenase-2 mRNA expression in the liver or reduction of serum total cholesterol levels. These results indicate that age may play a significant role in the efficacy of chemoprevention of colon cancer by curcumin.

Age-related differential responses to curcumin-induced apoptosis during the initiation of colon cancer in rats

Curcumin is a widely-used dietary supplement and a chemopreventive agent for various cancers. Pre-clinical chemopreventive studies rarely consider the effect of aging. We previously reported that unlike young animals, curcumin is ineffective in middle-aged rats for colon chemoprevention. This study investigated whether resistance to apoptosis during cancer initiation contributes to this age-dependent effect. Young, middle-aged, and old F344 rats were fed either curcumin (0.6%) or control diet. Colonic apoptosis was evaluated 0, 8, and 16 h after azoxymethane (AOM) injection. Colonic Hsp70 mRNA levels, caspase-9 activity, cell proliferation, and crypt morphology were measured. In AOM-treated rats, only middle-aged rats were resistant to curcumin-induced apoptosis whereas cell proliferation was reduced by curcumin in all ages. Curcumin-induced apoptosis was mediated by caspase-9 in young but not older rats. Transcriptional Hsp70 expression was induced in only young rats and was suppressed by curcumin. Therefore, the age-related difference in curcumin chemoprevention is due to a differential response in induction of apoptosis. The mitochondria-dependent pathway seems to mediate curcumin-induced apoptosis in young but not older animals. Hsp70 expression was not related with resistance to curcumin-induced apoptosis. Understanding age-related differences in the apoptotic response may lead to improved translation from pre-clinical animal studies to humans.

Effect of azoxymethane and curcumin on transcriptional levels of cyclooxygenase-1 and -2 during initiation of colon carcinogenesis

Background. Curcumin is well documented as an effective colonic chemopreventive agent in preclinical studies. Inhibition of arachidonic acid metabolism has been considered one of anticarcinogenic mechanisms of curcumin. We recently reported resistance of middle-aged F344 male rats to inhibition of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) by curcumin (Nutr Cancer, 48, 37–43). It was important to confirm this finding and to find potential mechanisms responsible, as loss of preventive activity of curcumin due to aging was a novel finding, with important implications for human intervention trials. Methods. To confirm our previous findings, and investigate arachidonic acid metabolism as a potential mechanism of age-related differences in response to curcumin, middle-aged F344 male rats were given AOM injections after being fed their experimental diets, 0.6% curcumin or control diet. Colonic ACF were evaluated and colonic levels of cyclooxygenase (COX)-1 and 2 mRNA and prostaglandin E2 (PGE2) were measured. Next, we investigated the short-term effect of AOM and curcumin on arachidonic acid metabolism in young rats. Six week-old rats were given injections of either AOM or untreated following their experimental diets. Colonic COX-1 and COX-2 mRNA as well as PGE2 levels were measured shortly after AOM treatment. Lastly, three different ages of F344 rats were treated with either AOM or saline, and colonic COX-1 and COX-2 mRNA levels were measured shortly after the injections to find if aging alters the effect of AOM on COX mRNA expression. Results. In middle-aged rats, dietary curcumin did not reduce the number of ACF and surprisingly increased colonic levels of COX-2 mRNA. Colonic COX-2 and PGE2 levels were also significantly increased in young rats fed curcumin after AOM injections. Interestingly, AOM did not affect COX-2 but decreased COX-1 expression in all ages. Conclusions. Our study suggests that during initiation, AOM inhibits colonic COX-1 expression without affecting COX-2 and dietary curcumin may increase COX-2 expression to compensate AOM-induced reduction of COX-1 expression.

Aging alters acute apoptotic response to azoxymethane in the colon of rats

Deregulation of apoptosis seems to contribute to the aging process in post-mitotic tissues. However, the effect of aging on regulation of apoptosis in the colon is largely unknown. We induced colonic apoptosis using azoxymethane (AOM) in three different aged (6 week, 12 month, and 22 month) F344 rats and assessed age-related differences in induction and potential molecular mechanisms of apoptosis. The incidence of colonic apoptosis was measured at 0, 4, 8, 16, and 24h after the AOM injection. Changes in transcriptional levels of NF-kappaB- and p53-regulated genes were measured following AOM exposure. Changes in colonic morphology were evaluated by measuring crypt proximity. Maximum apoptosis occurred at 8h after AOM injections in all age groups. However, apoptotic incidence was two- to threefold higher at the apoptotic peak in old compared to young rats. Bcl-xL, Bcl-2, and IAP-2 mRNA levels were down-regulated in young and old rats but stable in middle-aged rats after AOM injections. Transcriptional levels of Bax were not affected either by age or AOM. Expression of p21 was induced only in AOM-treated young rats. Crypt proximity was reduced in the older rats regardless of AOM treatment compared to young AOM-untreated rats. Our study suggests that older animals overly activate the apoptotic response to AOM and have modified colonic morphology. The precise mechanism(s) responsible for the over-active apoptotic response in older animals and its biological significance in relationship to loss of crypt architecture need to be further investigated.

Critical review of the current literature on the safety of sucralose

Sucralose is a non-caloric high intensity sweetener that is approved globally for use in foods and beverages. This review provides an updated summary of the literature addressing the safety of use of sucralose. Studies reviewed include chemical characterization and stability, toxicokinetics in animals and humans, assessment of genotoxicity, and animal and human feeding studies. Endpoints evaluated include effects on growth, development, reproduction, neurotoxicity, immunotoxicity, carcinogenicity and overall health status. Human clinical studies investigated potential effects of repeated consumption in individuals with diabetes. Recent studies on the safety of sucralose focused on carcinogenic potential and the effect of sucralose on the gut microflora are reviewed. Following the discovery of sweet taste receptors in the gut and studies investigating the activation of these receptors by sucralose lead to numerous human clinical studies assessing the effect of sucralose on overall glycemic control. Estimated daily intakes of sucralose in different population subgroups, including recent studies on children with special dietary needs, consistently find that the intakes of sucralose in all members of the population remain well below the acceptable daily intake. Collectively, critical review of the extensive database of research demonstrates that sucralose is safe for its intended use as a non-caloric sugar alternative.