Comparison of the effect of raw and blanched-frozen broccoli on DNA damage in colonocytes.

Consumption of cruciferous vegetables may protect against colorectal cancer. Cruciferous vegetables are rich in a number of bioactive constituents including polyphenols, vitamins and glucosinolates. Before consumption, cruciferous vegetables often undergo some form of processing that reduces their content of bioactive constituents and may determine whether they exert protective effects. The aim of this study was to compare the ability of raw and blanched-frozen broccoli to protect colonocytes against DNA damage, improve antioxidant status and induce xenobiotic metabolizing enzymes (XME). Fifteen Landrace × Large White male pigs were divided into five age-matched and weight-matched sets (79 days, SD 3, and 34·7 kg, SD 3·9, respectively). Each set consisted of siblings to minimize genetic variation. Within each set, pigs received a cereal-based diet, unsupplemented (control) or supplemented with 600 g day(-1) of raw or blanched-frozen broccoli for 12 days. The consumption of raw broccoli caused a significant 27% increase in DNA damage in colonocytes (p = 0·03) relative to the control diet, whereas blanched-frozen broccoli had no significant effect. Both broccoli diets had no significant effect on plasma antioxidant status or hepatic and colonic XME. This study is the first to report that the consumption of raw broccoli can damage DNA in porcine colonocytes.

Measuring the difference between actual and reported food intakes in the context of energy balance under laboratory conditions.

To date, no study has directly and simultaneously measured the discrepancy between what people actually eat and what they report eating under observation in the context of energy balance (EB). The present study aimed to objectively measure the 'extent' and 'nature' of misreporting of dietary intakes under conditions in which EB and feeding behaviour were continuously monitored. For this purpose, a total of fifty-nine adults were recruited for 12 d, involving two 3 d overt phases and two 3 d covert phases of food intake measurement in a randomised cross-over design. Subjects had ad libitum access to a variety of familiar foods. Food intake was covertly measured using a feeding behaviour suite to establish actual energy and nutrient intakes. During the overt phases, subjects were instructed to self-report food intake using widely accepted methods. Misreporting comprised two separate and synchronous phenomena. Subjects decreased energy intake (EI) when asked to record their food intake (observation effect). The effect was significant in women ( - 8 %, P< 0·001) but not in men ( - 3 %, P< 0·277). The reported EI was 5 to 21 % lower (reporting effect) than the actual intake, depending on the reporting method used. Semi-quantitative techniques gave larger discrepancies. These discrepancies were identical in men and women and non-macronutrient specific. The 'observation' and 'reporting' effects combined to constitute total misreporting, which ranged from 10 to 25 %, depending on the intake measurement assessed. When studied in a laboratory environment and EB was closely monitored, subjects under-reported their food intake and decreased the actual intake when they were aware that their intake was being monitored.

Effect of inulin on the human gut microbiota: stimulation of Bifidobacterium adolescentis and Faecalibacterium prausnitzii.

Prebiotics are food ingredients that improve health by modulating the colonic microbiota. The bifidogenic effect of the prebiotic inulin is well established; however, it remains unclear which species of Bifidobacterium are stimulated in vivo and whether bacterial groups other than lactic acid bacteria are affected by inulin consumption. Changes in the faecal microbiota composition were examined by real-time PCR in twelve human volunteers after ingestion of inulin (10 g/d) for a 16-d period in comparison with a control period without any supplement intake. The prevalence of most bacterial groups examined did not change after inulin intake, although the low G+C % Gram-positive species Faecalibacterium prausnitzii exhibited a significant increase (10.3% for control period v. 14.5% during inulin intake, P=0.019). The composition of the genus Bifidobacterium was studied in four of the volunteers by clone library analysis. Between three and five Bifidobacterium spp. were found in each volunteer. Bifidobacterium adolescentis and Bifidobacterium longum were present in all volunteers, and Bifidobacterium pseudocatenulatum, Bifidobacterium animalis, Bifidobacterium bifidum and Bifidobacterium dentium were also detected. Real-time PCR was employed to quantify the four most prevalent Bifidobacterium spp., B. adolescentis, B. longum, B. pseudocatenulatum and B. bifidum, in ten volunteers carrying detectable levels of bifidobacteria. B. adolescentis showed the strongest response to inulin consumption, increasing from 0.89 to 3.9% of the total microbiota (P=0.001). B. bifidum was increased from 0.22 to 0.63% (P<0.001) for the five volunteers for whom this species was present.

Influence of cooking duration of cabbage and presence of colonic microbiota on the excretion of N-acetylcysteine conjugates of allyl isothiocyanate and bioactivity of phase 2 enzymes in F344 rats.

Isothiocyanates have been implicated in the cancer-protective effects of brassica vegetables. When cabbage is consumed, sinigrin is hydrolysed by plant or microbial myrosinase partly to allyl isothiocyanate (AITC), which is mainly excreted as N-acetylcysteine conjugates (NAC) of AITC in urine. The effect of cooking cabbage on the excretion of NAC of AITC, and glutathione-S-transferase (GST) and uridine 5'-diphospho-glucuronosyl transferase (UGT) activity in rat liver and colon was investigated. Germ-free (GF) and human faecal microbiota-associated (HFM) rats were fed a control diet containing 20 % raw, lightly cooked, or fully cooked cabbage for 14 d. When plant myrosinase was present, excretion of NAC of AITC/24 h was increased by 1.4 and 2.5 times by the additional presence of microbial myrosinase after consumption of raw and lightly cooked cabbage respectively. When plant myrosinase was absent, as after consumption of fully cooked cabbage, excretion of the AITC conjugate was almost zero in GF and HFM rats. None of the cabbage diets modified hepatic GST activity. When microbiota was absent, colonic GST was 1.3-fold higher after fully cooked cabbage, and hepatic UGT was increased by 1.4-1.8-fold after all cabbage diets, compared with the control feed. There were no differences in GST or UGT following cabbage consumption when microbiota was present. It is possible that other constituents of cabbage, rather than metabolites of glucosinolates per se, may be responsible for changes in phase 2 enzyme activity. The main effect of cooking cabbage and altering colonic microbiota was on excretion of NAC of AITC.

Influence of cabbage processing methods and prebiotic manipulation of colonic microflora on glucosinolate breakdown in man.

Glucosinolate consumption from brassica vegetables has been implicated in reduction of cancer risk. The isothiocyanate breakdown products of glucosinolates appear to be particularly important as chemoprotective agents. Before consumption, brassica vegetables are generally cooked, causing the plant enzyme, myrosinase, to be denatured, influencing the profile of glucosinolate breakdown products produced. Some human intestinal microflora species show myrosinase-like activity (e.g. bifidobacteria). We aimed to increase bifidobacteria by offering a prebiotic (inulin) in a randomised crossover study. Six volunteers consumed inulin (10 g/d) for 21 d followed by a 21 d control period (no inulin). Treatment periods were reversed for the remaining six volunteers. During the last 5 d of each period two cabbage-containing meals were consumed. Total urine output was collected for 24 h following each meal. Cabbage was microwaved for 2 min (lightly cooked) or 5.5 min (fully cooked). Faecal samples were collected at the start and after the inulin and control treatments. Bifidobacteria were enumerated by real-time PCR. Allyl isothiocyanate production was quantified by measuring urinary excretion of allyl mercapturic acid (AMA). Bifidobacteria increased following prebiotic supplementation (P < 0.001) but there was no impact of this increase on AMA excretion. AMA excretion was greater following consumption of lightly cooked cabbage irrespective of prebiotic treatment (P < 0.001). In conclusion, the most effective way to increase isothiocyanate production may be to limit the length of time that brassica vegetables are cooked prior to consumption.

Effect of cooking brassica vegetables on the subsequent hydrolysis and metabolic fate of glucosinolates.

The protective effects of brassica vegetables against cancer may be partly related to their glucosinolate content. Glucosinolates are hydrolysed by plant myrosinase following damage of plant tissue. Isothiocyanates are one of the main groups of metabolites of glucosinolates and are implicated in the preventive effect against cancer. During cooking of brassica the glucosinolate-myrosinase system may be modified as a result of inactivation of plant myrosinase, loss of enzymic cofactors such as epithiospecifier protein, thermal breakdown and/or leaching of glucosinolates and their metabolites or volatilisation of metabolites. Cooking brassica affects the site of release of breakdown products of glucosinolates, which is the upper gastrointestinal tract following consumption of raw brassica containing active plant myrosinase. After consumption of cooked brassica devoid of plant myrosinase glucosinolates are hydrolysed in the colon under the action of the resident microflora. Feeding trials with human subjects have shown that hydrolysis of glucosinolates and absorption of isothiocyanates are greater following ingestion of raw brassica with active plant myrosinase than after consumption of the cooked plant with denatured myrosinase. The digestive fate of glucosinolates may be further influenced by the extent of cell rupture during ingestion, gastrointestinal transit time, meal composition, individual genotype and differences in colonic microflora. These sources of variation may partly explain the weak epidemiological evidence relating consumption of brassica to prevention against cancer. An understanding of the biochemical changes occurring during cooking and ingestion of brassica may help in the design of more robust epidemiological studies to better evaluate the protective effects of brassica against cancer.

Effect of meal composition and cooking duration on the fate of sulforaphane following consumption of broccoli by healthy human subjects.

The isothiocyanate, sulforaphane, has been implicated in the cancer-protective effects of brassica vegetables. When broccoli is consumed, sulforaphane is released from hydrolysis of glucoraphanin by plant myrosinase and/or colonic microbiota. The influence of meal composition and broccoli-cooking duration on isothiocyanate uptake was investigated in a designed experiment. Volunteers (n 12) were each offered a meal, with or without beef, together with 150 g lightly cooked broccoli (microwaved 2.0 min) or fully cooked broccoli (microwaved 5.5 min), or a broccoli seed extract. They received 3 g mustard containing pre-formed allyl isothiocyanate (AITC) with each meal. Urinary output of allyl (AMA) and sulforaphane (SFMA) mercapturic acids, the biomarkers of production of AITC and sulforaphane respectively, were measured for 24 h after meal consumption. The estimated yield of sulforaphane in vivo was about 3-fold higher after consumption of lightly cooked broccoli than fully cooked broccoli. Absorption of AITC from mustard was about 1.3-fold higher following consumption of the meat-containing meal compared with the non meat-containing alternative. The meal matrix did not significantly influence the hydrolysis of glucoraphanin and its excretion as SFMA from broccoli. Isothiocyanates may interact with the meal matrix to a greater extent if they are ingested pre-formed rather than after their production from hydrolysis of glucosinolates in vivo. The main influence on the production of isothiocyanates in vivo is the way in which brassica vegetables are cooked, rather than the effect of the meal matrix.

Changes in glucosinolate concentrations, myrosinase activity, and production of metabolites of glucosinolates in cabbage (Brassica oleracea Var. capitata) cooked for different durations.

In cabbage, glucosinolates such as sinigrin are hydrolyzed by plant myrosinase to allyl isothiocyanate (AITC), allyl cyanide, and, in the presence of an epithiospecifier protein, 1-cyano-2,3-epithiopropane (CEP). Isothiocyanates have been implicated in the cancer-protective effects of Brassica vegetables. The effect of processing on the hydrolysis of glucosinolates was investigated in cabbage. Cabbage was steamed or microwaved for six time durations over 7 min. Glucosinolate concentrations were slightly reduced after microwave cooking (P < 0.001) but were not influenced after steaming (P < 0.05). Myrosinase activity was effectively lost after 2 min of microwave cooking and after 7 min of steaming. Hydrolysis of residual glucosinolates following cooking yielded predominantly CEP at short cooking durations and AITC at longer durations until myrosinase activity was lost. Lightly cooked cabbage produced the highest yield of AITC on hydrolysis in vitro, suggesting that cooking Brassica vegetables for a relatively short duration may be desirable from a health perspective.

Cruciferous vegetables and colo-rectal cancer.

Cruciferous vegetables have been studied extensively for their chemoprotective effects. Although they contain many bioactive compounds, the anti-carcinogenic actions of cruciferous vegetables are commonly attributed to their content of glucosinolates. Glucosinolates are relatively biologically inert but can be hydrolysed to a range of bioactive compounds such as isothiocyanates (ITC) and indoles by the plant-based enzyme myrosinase, or less efficiently by the colonic microflora. A number of mechanisms whereby ITC and indoles may protect against colo-rectal cancer have been identified. In experimental animals cruciferous vegetables have been shown to inhibit chemically-induced colon cancer. However, the results of recent epidemiological cohort studies have been inconsistent and this disparity may reflect a lack of sensitivity of such studies. Possible explanations for the failure of epidemiological studies to detect an effect include: assessment of cruciferous vegetable intake by methods that are subject to large measurement errors; the interaction between diet and genotype has not been considered: the effect that post-harvest treatments may have on biological effects of cruciferous vegetables has not been taken into account.

Synthesis and biological activities of bisnaphthalimido polyamines derivatives: cytotoxicity, DNA binding, DNA damage and drug localization in breast cancer MCF 7 cells.

New bisoxynaphthalimidopolyamines (BNIPOPut, BNIPOSpd and BNIPOSpm) were synthesized. Their cytotoxic properties were evaluated against breast cancer MCF 7 cells and compared with bisnaphthalimidopolyamines BNIPSpd and BNIPSpm. Among the bisoxynaphthalimido polyamines, BNIPOSpm and BNIPOSpd exhibited cytotoxic activity with an IC50 f 29.55 and 27.22 microM, respectively, while BNIPOPut failed to exert significant cytotoxicity after 48-h drug exposure. DNA binding was determined by midpoint of thermal denaturation (Tm) measurement, ethidium bromide displacement and DNA gel mobility. Both BNIPOSpm and BNIPOSpd exhibited strong binding affinities with DNA. BNIPOPut had the least effect. The results were compared with other cytotoxic bisnaphthalimido compounds (BNIPSpm and BNIPSpd) previously reported by us. Using the single cell gel electrophoresis assay, it was found that BNIPSpm and BNIPSpd caused substantial DNA damage to MCF 7 treated cells while BNIPOSpm showed no significant effect over a range of drug concentrations after 4-h drug exposure. However, after 12-h drug exposure, BNIPOSpm had induced significant DNA damage similar to that of BNIPSpm (after 4-h drug exposure). Fluorescence microscopic analysis revealed that at 1 microM drug concentration and after 6-h drug exposure, both BNIPSpm and BNIPSpd were located within the cell while the presence of BNIPOSpm, was not observed. Therefore, we conclude that BNIPSpd, BNIPSpm and BNIPOSpm induce DNA damage consistent with their rate of uptake into the cells.