Modulation of detoxification enzymes by watercress: in vitro and in vivo investigations in human peripheral blood cells.

BACKGROUND: Epidemiological studies indicate that consumption of cruciferous vegetables (CV) can reduce the risk of cancer. Supposed mechanisms are partly the inhibition of phase I and the induction of phase II enzymes. AIM: The aim of this study was to investigate in vitro and in vivo effects of watercress (WC), a member of the CV family, on chemopreventive parameters using human peripheral blood mononuclear cells (PBMC) as surrogate cells. We investigated the hypothesis that WC reduces cancer risk by inducing detoxification enzymes in a genotype-dependent manner. METHODS: In vitro gene expression and enzyme activity experiments used PBMC incubated with a crude extract from fresh watercress (WCE, 0.1-10 microL/mL with 8.2 g WC per 1 mL extract) or with one main key compound phenethyl isothiocyanate (PEITC, 1-10 microM). From an in vivo perspective, gene expression and glutathione S-transferase (GST) polymorphisms were determined in PBMC obtained from a human intervention study in which subjects consumed 85 g WC per day for 8 weeks. The influence of WC consumption on gene expression was determined for detoxification enzymes such as superoxide dismutase 2 (SOD2) and glutathione peroxidase 1 (GPX1), whilst the SOD and GPX activities in red blood cells were also analysed with respect to GST genotypes. RESULTS: In vitro exposure of PBMC to WCE or PEITC (24 h) increased gene expression for both detoxification enzymes GPX1 (5.5-fold, 1 microL/mL WCE, 3.7-fold 1 microM PEITC) and SOD2 (12.1-fold, 10 microL/mL WCE, 7.3-fold, 10 microM PEITC), and increased SOD2 activity (1.9-fold, 10 microL/mL WCE). The WC intervention had no significant effect on in vivo PBMC gene expression, as high individual variations were observed. However, a small but significant increase in GPX (p = 0.025) and SOD enzyme activity (p = 0.054) in red blood cells was observed in GSTM1*0, but not in GSTM1*1 individuals, whilst the GSTT1 genotype had no impact. CONCLUSION: The results indicate that WC is able to modulate the enzymes SOD and GPX in blood cells in vitro and in vivo, and suggest that the capacity of moderate intake of CV to induce detoxification is dependent in part on the GSTM1 genotype.

Anthelmintic efficacy of extracts of Spigelia anthelmia Linn on experimental Nippostrongylus braziliensis in rats.

Spigelia anthelmia Linn is used as a herb and is a common annual weed of cultivation in open re-growths, on unused land in towns as well as on road sides. The plant can grow to approximately 30 cm in height. The aim of this study was to screen extracts of Spigelia anthelmia for their anthelmintic activity against an experimental Nippostrongylus braziliensis infection in rats. Acute oral toxicity occurred at a dose of 1,140 mg/kg, while anthelmintic trials against Nippostrongylus braziliensis in rats using the aqueous fraction showed a progressive decrease in worm count with increasing dose (10, 13, 16, 20 and 25 mg per kg body weight) (p < 0.05). At 25 mg per kg body weight, the worm count was significantly lower than that at 10 mg per kg body weight (p < 0.05).

Relationship between dietary-induced changes in intestinal commensal microflora and duodenojejunal myoelectric activity monitored by radiotelemetry in the rat in vivo.

Interdigestive intestinal motility, and especially phase III of the migrating myoelectric/motor complex (MMC), is responsible for intestinal clearance and plays an important role in prevention of bacterial overgrowth and translocation in the gut. Yet previous results from gnotobiotic rats have shown that intestinal microflora can themselves affect the characteristics of the myoelectric activity of the gut during the interdigestive state. Given that the composition of the intestinal microflora can be altered by dietary manipulations, we investigated the effect of supplementation of the diet with synbiotics on intestinal microflora structure and the duodenojejunal myoelectric activity in the rat. To reduce animal distress caused by restraint and handling, which can itself affect GI motility, we applied radiotelemetry for duodenojejunal EMG recordings in conscious, freely moving rats. Thirty 16-month-old Spraque-Dawley rats were used. The diet for 15 rats (E group) was supplemented with chicory inulin, Lactobacillus rhamnosus and Bifidobacterium lactis. The remaining 15 rats were fed control diet without supplements (C group). Three rats from each group were implanted with three bipolar electrodes positioned at 2, 14 and 28 cm distal to the pylorus. After recovery, two 6 h recordings of duodenojejunal EMG were carried out on each operated rat. Subsequently, group C rats received feed supplements and group E rats received only control diet for 1 week, and an additional two 6 h recordings were carried out on each of these rats. Non-operated C and E rats were killed and samples of GI tract were collected for microbiological analyses. Supplementation of the diet with the pro- and prebiotics mixture increased the number of bifidobacteria, whereas it decreased the number of enterobacteria in jejunum, ileum, caecum and colon. In both caecum and colon, the dietary supplementation increased the number of total anaerobes and lactobacilli. Treatment with synbiotics increased occurrence of phase III of the MMC at all three levels of the small intestine. The propagation velocity of phase III in the whole recording segment was also increased from 3.7 +/- 0.2 to 4.4 +/- 0.2 cm min(-1) by dietary treatment. Treatment with synbiotics increased the frequency of response potentials of the propagated phase III of the MMC at both levels of the jejunum, but not in the duodenum. In both parts of the jejunum, the supplementation of the diet significantly decreased the duration of phase II of the MMC, while it did not change the duration of phase I and phase III. Using the telemetry technique it was demonstrated that changes in the gastrointestinal microflora exhibited an intestinal motility response and, more importantly, that such changes can be initiated by the addition of synbiotics to the diet.

Anthelmintic efficacy of extracts of Spigelia anthelmia Linn on experimental Nippostrongylus braziliensis in rats

Spigelia anthelmia Linn is used as a herb and is a common annual weed of cultivation in open re-growths, on unused land in towns as well as on road sides. The plant can grow to approximately 30 cm in height. The aim of this study was to screen extracts of Spigelia anthelmia for their anthelmintic activity against an experimental Nippostrongylus braziliensis infection in rats. Acute oral toxicity occurred at a dose of 1,140 mg/kg, while anthelmintic trials against Nippostrongylus braziliensis in rats using the aqueous fraction showed a progressive decrease in worm count with increasing dose (10, 13, 16, 20 and 25 mg per kg body weight) (p < 0.05). At 25 mg per kg body weight, the worm count was significantly lower than that at 10 mg per kg body weight (p < 0.05).

Monitoring temozolomide treatment of low-grade glioma with proton magnetic resonance spectroscopy.

Assessment of low-grade glioma treatment response remains as much of a challenge as the treatment itself. Proton magnetic resonance spectroscopy ((1)H-MRS) and imaging were incorporated into a study of patients receiving temozolomide therapy for low-grade glioma in order to evaluate and monitor tumour metabolite and volume changes during treatment. Patients (n=12) received oral temozolomide (200 mg m(-2) day(-1)) over 5 days on a 28-day cycle for 12 cycles. Response assessment included baseline and three-monthly magnetic resonance imaging studies (pretreatment, 3, 6, 9 and 12 months) assessing the tumour size. Short (TE (echo time)=20 ms) and long (TE=135 ms) echo time single voxel spectroscopy was performed in parallel to determine metabolite profiles. The mean tumour volume change at the end of treatment was -33% (s.d.=20). The dominant metabolite in long echo time spectra was choline. At 12 months, a significant reduction in the mean choline signal was observed compared with the pretreatment (P=0.035) and 3-month scan (P=0.021). The reduction in the tumour choline/water signal paralleled tumour volume change and may reflect the therapeutic effect of temozolomide.

Isoflavone phytoestrogens consumed in soy decrease F(2)-isoprostane concentrations and increase resistance of low-density lipoprotein to oxidation in humans.

BACKGROUND: Oxidative damage to lipids may be involved in the etiology of atherosclerosis, cardiovascular disease in general, and cancer. The soy isoflavone phytoestrogens, genistein and daidzein, and equol (a daidzein metabolite produced by intestinal microflora) are antioxidants in vitro; equol is a particularly good inhibitor of LDL oxidation and membrane lipid peroxidation. OBJECTIVE: We sought to investigate the effects of a diet enriched with soy containing isoflavones on in vivo biomarkers of lipid peroxidation and resistance of LDL to oxidation, compared with a diet enriched with soy from which the isoflavones had been extracted. DESIGN: : A randomized, crossover design was used to compare diets enriched with soy that was low or high in isoflavones in 24 subjects. Plasma concentrations of an F(2)-isoprostane, 8-epi-prostaglandin F(2)(alpha) (8-epi-PGF(2)(alpha)), a biomarker of in vivo lipid peroxidation, and resistance of LDL to copper-ion-induced oxidation were determined. RESULTS: Plasma concentrations of 8-epi-PGF(2)(alpha) were significantly lower after the high-isoflavone dietary treatment than after the low-isoflavone dietary treatment (326 +/- 32 and 405 +/- 50 ng/L, respectively; P = 0.028) and the lag time for copper-ion-induced LDL oxidation was longer (48 +/- 2.4 and 44 +/- 1.9 min, respectively; P = 0.017). Lag time for oxidation of unfractionated plasma and plasma concentrations of malondialdehyde, LDL alpha-tocopherol, polyunsaturated fatty acids, and isoflavonoids did not differ significantly between dietary treatments. CONCLUSIONS: Consumption of soy containing naturally occurring amounts of isoflavone phytoestrogens reduced lipid peroxidation in vivo and increased the resistance of LDL to oxidation. This antioxidant action may be significant with regard to risk of atherosclerosis, cardiovascular disease in general, and cancer.

Effects of soy or rye supplementation of high-fat diets on colon tumour development in azoxymethane-treated rats.

Evidence is accumulating that a diet high in plant-derived foods may be protective against cancer. One class of plant component under increasing investigation is the phytoestrogens of which there are two main groups: the isoflavones, found mainly in soy products, and the lignans, which are more ubiquitous and are found in fruit, vegetables and cereals with high levels being found in flaxseed. In this study, we have used carefully balanced high-fat (40% energy) diets: a control diet (containing low isoflavone soy protein as the sole protein source), a rye diet (the control diet supplemented with rye bran) and a soy diet (containing as protein source a high isoflavone soy protein). The effect of these diets on the development of colonic cancer was studied in F-344 rats treated with the carcinogen, azoxymethane (two doses of 15 mg/kg given 1 week apart). Colons from treated animals were examined for aberrant crypt foci (ACF) and tumours after 12 and 31 weeks. Results after 12 weeks showed no differences in the total number of ACF in the control, soy or rye bran groups. However, the soy group had increased numbers of small ACF (less than four crypts/focus) while the rye group had decreased numbers of large ACF (greater than six crypts/focus). Examination of colons after 31 weeks gave similar low numbers of ACF in each group with no differences in multiplicity. There were no differences in the number of tumours between the control (1.36 tumours/rat) and soy (1.38 tumours/rat) groups. However, there was a significant decrease in the number of tumours in the rye group (0.17 tumours/rat). These results suggest that soy isoflavones have no effect on the frequency of colonic tumours in this model while rye bran supplementation decreases the frequency of colon cancer. This effect is due not to a decrease in early lesions but in their progression to larger multi-crypt ACF. The study also supports the hypothesis that larger ACF are more predictive of subsequent tumorigenicity.

Effect of dietary arginine on urinary nitrate excretion in germ-free rats.

The effect of dietary arginine on the endogenous synthesis of nitrate in germ-free rats was investigated. The animals were fed, for up to 18 days, purified diets containing either casein or lactalbumin (proteins differing in arginine content) with or without additional free arginine. Urine was collected from all animals for 4 days, and the animals were then dosed ip with Escherichia coli lipopolysaccharide (LPS) to stimulate nitrate synthesis and urine collected for a further 4 days. The urine samples were analysed for nitrate by ion chromatography. Although the excretion of nitrate was markedly stimulated by LPS treatment, there were no significant effects of protein source or arginine supplementation of the diet.

The use of rats associated with a human faecal flora as a model for studying the effects of diet on the human gut microflora.

The activities of four bacterial biotransformation enzymes (beta-glucosidase, beta-glucuronidase, nitrate reductase and nitroreductase) were measured in the caecal contents of conventional flora rats or germ-free rats contaminated with a mixed, human faecal flora and compared with activities present in a fresh human stool preparation. Both the conventional flora rats and the rats inoculated with a human flora exhibited an enzyme profile generally similar to that of human faeces, although the conventional rat flora exhibited negligible nitrate reductase activity. The enzyme profile remained essentially unaltered in both human flora preparations following supplementation of the diet with pectin, whereas the conventional rat flora responded to this plant cell wall carbohydrate with a significant increase in nitrate reductase activity. The results demonstrate that enzymic activities of the human faecal microflora can be simulated in rats associated with a mixed population of human intestinal bacteria.

Dietary modification of intestinal bacterial enzyme activities--potential formation of toxic agents in the gut.

The bacterial population colonising the large intestine is able to metabolise a variety of ingested or endogenously produced substances to products, some of which possess toxic, mutagenic or carcinogenic properties. Dietary components, resistant to digestion and absorption in the upper alimentary tract, may influence these reactions by altering the environment of the gut or through the provision of nutrients to the flora. Evidence for the involvement of bacterial enzymes in the formation of toxic products in vivo has come largely from animal studies, particularly where fermentable plant cell-wall components are present in the diet. The role of diet in the modification of toxicologically important bacterial biotransformation processes will be discussed. Preliminary data will also be presented from a study demonstrating changes in the enzymic activity of the human faecal flora induced by pectin and bran. The significance of these changes to the disposition of chemicals in the gut will be discussed.

Effect of mixtures of dietary fibres on the enzyme activity of the rat caecal microflora.

The enzyme activity of the caecal microflora from weanling rats was determined after feeding 1 of 3 basal diets (purified fibre-free; purified plus cellulose; and stock), with or without additional dietary fibre (pectin, i-carrageenan or carboxymethylcellulose 5% w/w). The wet weight of caecal contents and total bacterial numbers were similar for the purified fibre-free and purified plus cellulose diets, yet were significantly higher in animals fed the stock diet. Pectin supplementation of the basal diets had no effect of caecal bacterial numbers, but significantly increased total nitrate reductase activity per caecum except when added to stock diet. Carrageenan decreased caecal bacterial numbers and most enzyme activities with both purified diets, and to a lesser extent with the stock diet. Carboxymethylcellulose increased bacterial numbers and enzyme activities, particularly beta-glucosidase and nitrate reductase when added to the purified diet but not when added to either the purified diet plus cellulose or the stock diet. The results demonstrate that the effects of dietary fibre components on the rat caecal microflora are dependent upon the initial fibre content of the diet base.

Effects of diet on mercury metabolism and excretion in mice given methylmercury: role of gut flora.

Mice fed either (1) a pelleted rodent diet, (2) evaporated milk, or (3) a synthetic diet (high protein, low fat) exhibited different rates of whole body mercury elimination and fecal mercury excretion after exposure (per os) to methylmercuric chloride. The percentage of the total mercury body burden present as mercuric mercury was highest (35.3%) in mice fed the synthetic diet (which had the highest rate of mercury elimination) and lowest (6.6%) in the animals having the lowest mercury elimination rate (milk-fed mice). Mice fed the synthetic diet had lower mercury concentrations and had a higher proportion of mercuric mercury in their tissues than the mice from the other dietary groups. Treatment of the mice with antibiotics throughout the experimental period to suppress the gut flora reduced fecal mercury excretion and the dietary differences in whole body retention of mercury. Tissue mercury concentrations and proportion of organic mercury in feces, cecal contents, liver, and kidneys were increased by antibiotic treatment of mice fed the pelleted or synthetic diets. These results are consistent with the theory that demethylation of methylmercury by intestinal microflora is a major factor determining the excretion rate of mercury.

Hydrocolloid food additives and rat caecal microbial enzyme activities.

Agar, carboxymethylcellulose, carrageenan, guar gum, gum acacia, locust-beam gum or pectin (50 g/kg diet), given to weanling rats for 4 wk, increased the weight of the caecal wall and the caecal contents. Feeding carboxymethylcellulose, guar gum or pectin significantly increased, and feeding carrageenan decreased, the total bacterial population of the caecum. Feeding carboxymethylcellulose significantly increased in vitro activity of bacterial azoreductase, beta-glucosidase, beta-glucuronidase, nitrate reductase, nitroreductase and urease. Guar gum, gum acacia and locust-bean gum each increased at least three of these activities. In contrast, feeding carrageenan greatly decreased all microbial enzyme activities, while agar decreased beta-glucosidase, beta-glucuronidase and nitroreductase activities.

A comparison of the activity of five microbial enzymes in cecal content from rats, mice, and hamsters, and response to dietary pectin.

Rats, mice, and hamsters were fed a fiber-free purified diet for 30 days and the activity of a number of cecal microbial enzymes was determined. Expressed per gram cecal content, azoreductase activity was greatest in preparations from the hamster and least from the mouse, and beta-glucosidase and beta-glucuronidase activities were least active from the rat. Nitroreductase was less active and nitrate reductase more active from the hamster in comparison to the other species. When expressed per kilogram body weight, bacterial activities were always greatest from the hamster. When the basal diet was supplemented with pectin (50 g/kg diet), nitrate reductase activity was increased six- to sevenfold per gram cecal content for rats and mice (tenfold when expressed per kilogram body weight), but there was no effect on the nitrate reductase activity of hamster microflora. Pectin also significantly increased beta-glucuronidase activity in rats, but significantly reduced the activities of the other enzymes in at least one of the three species.

Effect of dietary carrageenan and pectin on the reduction of nitro-compounds by the rat caecal microflora.

Rats were fed either a basal purified diet, or that diet supplemented with 50 g/kg pectin or iota carrageenan for 50 days, and caecal microbial nitroreductase activity determined using p-nitrobenzoic acid, p-nitrophenol, 2,4-dinitrotoluene, nitrofurantoin and metronidazole as substrates. Both pectin and carrageenan increased the weight of caecal contents, and pectin also increased the number of bacteria per caecum. In contrast, carrageenan decreased the caecal bacterial population. Pectin significantly increased the rate of reduction of metronidazole and the rate of conversion of p-nitrobenzoic acid to p-aminobenzoic acid, while carrageenan significantly decreased the rate of reduction of every compound studied. The results demonstrate that microbial reduction of the nitro-group may be altered by diet, although the response found with one nitro-compound may differ from that seen with another substrate.

Interaction between pectin and rat hindgut microflora.

The contents of the lower alimentary tract from rats fed a semisynthetic, pectin-supplemented diet showed increased nitrate reductase activity and an increase in the amount of luminal contents in the intestine and cecum. Nitrate reductase activity was associated with the insoluble fraction of the gut contents which was sedimented by centrifugation (5,100 X g,20 min) and was abolished after treating the animals with streptomycin, neomycin, and bacitracin for 7 days. The pectin-dependent increase in cecal size and microbial nitrate reduction were reversed when animals were transferred from a pectin-supplemented onto a control semisynthetic diet. Polygalacturonic acid (pectic acid) was without effect on either cecal size or cecal microbial nitrate reductase activity. The studies demonstrate that pectin influences microbial metabolism in the alimentary tract.