Apiaceous and Cruciferous Vegetables Fed During the Post-Initiation Stage Reduce Colon Cancer Risk Markers in Rats.

BACKGROUND: Vegetable consumption reduces colon cancer risk when fed in the initiation stage of carcinogenesis; however, the effect of vegetable consumption during the post-initiation stage has rarely been examined. OBJECTIVE: We investigated the chemopreventive effects of feeding apiaceous and cruciferous vegetables on colon cancer risk in the post-initiation stage. METHODS: Thirty male Wistar rats (∼5 wk, 92 g) were subcutaneously injected with 1,2-dimethylhydrazine 1 time/wk for 2 wk. One week after the last dose, rats were randomly assigned to 3 groups: the basal diet, an apiaceous vegetable-containing diet (API; 21% fresh wt/wt), or a cruciferous vegetable-containing diet (CRU; 21% fresh wt/wt). All diets contained ∼20% protein, 7% fat, and 63% digestible carbohydrate. Experimental diets were fed for 10 wk, after which colons were harvested. RESULTS: CRU reduced aberrant crypt foci (ACF) number compared to the basal group (P = 0.014) and API (P = 0.013), whereas API decreased the proportion of dysplastic ACF relative to the basal group (P < 0.05). Both CRU and API reduced doublecortin-like kinase 1-positive marker expression relative to basal by 57.9% (P = 0.009) and 51.4% (P < 0.02). The numbers of CD44-positive ACF did not differ between the groups. We identified 14 differentially expressed microRNAs (miRNAs). Of these, expression of 6 miRNAs were greater or tended to be greater (P ≤ 0.10) in one or both vegetable-containing groups compared to the basal group. Bioinformatic analysis of these expression changes in miRNA predicted a change in WNT/ß-catenin signaling, indicating downregulation of ß-catenin in the vegetable-fed groups. Consistent with this bioinformatics analysis, ß-catenin-accumulated ACF were decreased in CRU (93.1%, P = 0.012), but not in API (54.4%, P = 0.125), compared to the basal group. CONCLUSION: Both apiaceous and cruciferous vegetables, fed post-initiation, reduce colonic preneoplastic lesions as well as cancer stem cell marker expression in rats, possibly by suppressing oncogenic signaling through changes in miRNA expression.

Apiaceous vegetable consumption decreases PhIP-induced DNA adducts and increases methylated PhIP metabolites in the urine metabolome in rats.

BACKGROUND: Heterocyclic aromatic amines, such as 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), are carcinogenic compounds produced during heating of protein-containing foods. Apiaceous vegetables inhibit PhIP-activating enzymes, whereas cruciferous vegetables induce both PhIP-activating and -detoxifying enzymes. OBJECTIVE: We investigated the effects of these vegetables, either alone or combined, on PhIP metabolism and colonic DNA adduct formation in rats. METHODS: Male Wistar rats were fed cruciferous vegetables (21%, wt:wt), apiaceous vegetables (21%, wt:wt), or a combination of both vegetables (10.5% wt:wt of each). Negative and positive control groups were fed an AIN-93G diet. After 6 d, all groups received an intraperitoneal injection of PhIP (10 mg · kg body weight(-1)) except for the negative control group, which received only vehicle. Urine was collected for 24 h after the injection for LC-tandem mass spectrometry metabolomic analyses. On day 7, rats were killed and tissues processed. RESULTS: Compared with the positive control, cruciferous vegetables increased the activity of hepatic PhIP-activating enzymes [39.5% and 45.1% for cytochrome P450 (CYP) 1A1 (P = 0.0006) and CYP1A2 (P < 0.0001), respectively] and of uridine 5'-diphospho-glucuronosyltransferase 1A (PhIP-detoxifying) by 24.5% (P = 0.0267). Apiaceous vegetables did not inhibit PhIP-activating enzymes, yet reduced colonic PhIP-DNA adducts by 20.4% (P = 0.0496). Metabolomic analyses indicated that apiaceous vegetables increased the relative abundance of urinary methylated PhIP metabolites. The sum of these methylated metabolites inversely correlated with colonic PhIP-DNA adducts (r = -0.43, P = 0.01). We detected a novel methylated urinary PhIP metabolite and demonstrated that methylated metabolites are produced in the human liver S9 fraction. CONCLUSIONS: Apiaceous vegetables did not inhibit the activity of PhIP-activating enzymes in rats, suggesting that the reduction in PhIP-DNA adducts may involve other pathways. Further investigation of the importance of PhIP methylation in carcinogen metabolism is warranted, given the inverse correlation of methylated PhIP metabolites with a biomarker of carcinogenesis and the detection of a novel methylated PhIP metabolite.

Cruciferous vegetables have variable effects on biomarkers of systemic inflammation in a randomized controlled trial in healthy young adults.

BACKGROUND: Isothiocyanates in cruciferous vegetables modulate signaling pathways critical to carcinogenesis, including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), a central regulator of inflammation. Glutathione S-transferase (GST) M1 and GSTT1 metabolize isothiocyanates; genetic variants may result in differences in biologic response. OBJECTIVE: The objective of this study was to test whether consumption of cruciferous or cruciferous plus apiaceous vegetables altered serum concentrations of interleukin (IL)-6, IL-8, C-reactive protein (CRP), tumor necrosis factor (TNF) α, and soluble TNF receptor (sTNFR) I and II, and whether this response was GSTM1/GSTT1 genotype dependent. METHODS: In a randomized crossover trial, healthy men (n = 32) and women (n = 31) aged 20-40 y consumed 4 14-d controlled diets: basal (vegetable-free), single-dose cruciferous (1xC) [7 g vegetables/kg body weight (BW)], double-dose cruciferous (2xC) (14 g/kg BW), and cruciferous plus apiaceous (carrot family) (1xC+A) vegetables (7 and 4 g/kg BW, respectively), with a 21-d washout period between each intervention. Urinary isothiocyanate excretion was also evaluated as a marker of systemic isothiocyanate exposure. Fasting morning blood and urine samples were collected on days 0 and 14 and analyzed. RESULTS: IL-6 concentrations were significantly lower on day 14 of the 2xC and 1xC+A diets than with the basal diet [-19% (95% CI: -30%, -0.1%) and -20% (95% CI: -31%, -0.7%), respectively]. IL-8 concentrations were higher after the 1xC+A diet (+16%; 95% CI: 4.2%, 35.2%) than after the basal diet. There were no effects of diet on CRP, TNF-α, or sTNFRI or II. There were significant differences between GSTM1-null/GSTT1+ individuals for several biomarkers in response to 1xC+A compared with basal diets (CRP: -37.8%; 95% CI: -58.0%, -7.4%; IL-6: -48.6%; 95% CI: -49.6%, -12.0%; IL-8: 16.3%; 95% CI: 6.7%, 57.7%) and with the 2xC diet compared with the basal diet (IL-8: -33.2%; 95% CI: -43.0%, -1.4%; sTNFRI: -7.5%; 95% CI: -12.7%, -2.3%). There were no significant reductions in biomarker concentrations in response to diet among GSTM1+/GSTT1+ or GSTM1-null/GSTT1-null individuals. Twenty-four-hour urinary isothiocyanate excretion was not associated with any of the inflammation markers overall; however, IL-6 was inversely associated with total isothiocyanate excretion in GSTM1-null/GSTT1-null individuals (ß = -0.12; 95% CI: -0.19, -0.05). CONCLUSIONS: In this young, healthy population, consumption of cruciferous and apiaceous vegetables reduced circulating IL-6; however, results for other biomarkers of inflammation were not consistent.

Development of selective comprehensive two-dimensional liquid chromatography with parallel first-dimension sampling and second-dimension separation--application to the quantitative analysis of furanocoumarins in apiaceous vegetables.

Various implementations of two-dimensional high-performance liquid chromatography are increasingly being developed and applied to the analysis of complex materials, including those encountered in the analysis of foods, beverages, and nutraceuticals. Previously, we introduced the concept of selective comprehensive two-dimensional liquid chromatography (sLC × LC) as a hybrid between the more conventional, but extreme opposite sampling modes of heartcutting (LC-LC) and fully comprehensive (LC × LC) 2D separation. The sLC × LC approach breaks the link between first dimension ((1)D) sampling time and second dimension ((2)D) analysis time that is faced in LC × LC and allows very rapid (as low as 1 s) sampling of highly efficient (1)D separations, while at the same time allowing efficient (2)D separations on the timescale of tens of seconds. In this paper, we improve upon our previous sLC × LC work by demonstrating the ability to perform the processes of (1)D sampling and (2)D separation in parallel. This significantly improves the flexibility of the technique and allows targeted analysis of analytes that elute close together in time in the (1)D separation. To demonstrate the value of this added capability, we have developed a sLC × LC method using multi-wavelength ultraviolet absorbance detection for the quantitative analysis of six target furanocoumarin compounds in extracts of celery, parsley, and parsnips. We show that (2)D separations of (1)D effluent containing the target compounds of interest reveal the presence of unanticipated interferent peaks that would otherwise compromise the quantitative accuracy of the method. We also demonstrate the application of the chemometric method iterative key set factor analysis with alternating least-squares to sLC × LC to mathematically resolve target compounds that are only slightly separated chromatographically but not sufficiently resolved for accurate quantitation.

Colon Cancer Risk of a Westernized Diet Is Reduced in Mice by Feeding Cruciferous or Apiaceous Vegetables at a Lower Dose of Carcinogen but Not a Higher Dose.

Western-style diets (WD) are associated with greater risk of colon cancer. Exposure to 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP), a food-borne carcinogen, is linked to increased colon cancer risk. In contrast, intake of apiaceous and cruciferous vegetables (APIs and CRUs) is associated with reduced risk. Here we evaluated effects of a WD alone or a WD containing API or CRU, relative to a purified diet (basal), on colon cancer risk in mice. All diets were fed at one of two concentrations of PhIP (100 or 400 ppm). The activity of the hepatic PhIP-activating enzyme, cytochrome P450 (CYP) 1A2, was examined at week 4 and colonic precancerous lesions (aberrant crypt foci, ACF) were enumerated at week 12. In low PhIP-fed groups, CYP1A2 activity was greater for CRU than all other groups, which did not differ from one another. WD had a significantly greater effect on the formation of ACF than the basal diet. In groups fed API or CRU, the ACF number was reduced to the level observed in the basal diet-fed group. In high PhIP-fed groups, all WD-based diets had greater CYP1A2 activity than the basal diet-fed group. Surprisingly, the basal diet group had more ACF than the WD group, and API and CRU groups did not differ from the WD alone group. Thus, at the lower dose of PhIP, the WD increased colon cancer risk in mice, compared to a purified diet, and APIs and CRUs reduced the risk of the WD. However, at the higher dose of PhIP, the enhancement of colon cancer risk by the WD was not evident, nor was the chemopreventive effect of these vegetables.

Apiaceous Vegetables and Cruciferous Phytochemicals Reduced PhIP-DNA Adducts in Prostate but Not in Pancreas of Wistar Rats.

We previously showed rats fed with apiaceous vegetables, but not with their putative chemopreventive phytochemicals, reduced colonic DNA adducts formed by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a dietary procarcinogen. We report here the effects of feeding apiaceous and cruciferous vegetables versus their purified predominant phytochemicals, either alone or combined, on prostate and pancreatic PhIP-DNA adduct formation. In experiment I, male Wistar rats received three supplemented diets: CRU (cruciferous vegetables), API (apiaceous vegetables), and CRU+API (both types of vegetables). In experiment II, rats received three diets supplemented with phytochemicals matched to their levels in the vegetables from experiment I: P + I (phenethyl isothiocyanate and indole-3-carbinol), FC (furanocoumarins; 5-methoxypsoralen, 8-methoxypsoralen, and isopimpinellin), and COMBO (P + I and FC combined). After 6 days of feeding, PhIP was injected (10 mg/kg body weight) and animals were killed on day 7. PhIP-DNA adducts were analyzed by LC-MS/MS. In prostate, PhIP-DNA adducts were reduced by API (33%, P < .05), P + I (45%, P < .001), and COMBO (30%, P < .01). There were no effects observed in pancreas. Our results suggest that fresh vegetables and purified phytochemicals lower PhIP-DNA adducts and may influence cancer risk.

Comparison of short- and long-term exposure effects of cruciferous and apiaceous vegetables on carcinogen metabolizing enzymes in Wistar rats.

Cruciferous and apiaceous vegetables may be chemopreventive due to their ability to modulate carcinogen-metabolizing enzymes but whether the effects on such enzymes are sustained over time is unknown. To examine the short- and long-term effects of the vegetables, rats were fed one of four diets for 7, 30, or 60 d: AIN-93G, CRU (21% cruciferous vegetables-fresh broccoli, green cabbage, watercress), API (9% apiaceous vegetables - fresh parsnips, celery), or API + CRU (10.5% CRU + 4.5% API). Although CRU increased activity and protein expression of cytochrome P450 (CYP) 1A1 and CYP1A2 after 7 d, only activity was sustained after 30 and 60 d. There was a trend towards an interaction between the length of feeding period and CRU for CYP1A1 activity; activity increased with greater time of feeding. API increased CYP1A2 activity but decreased sulfotransferase 1A1 activity after 7 d, although not at later times. Altogether, increased CYP1A activity by CRU was maintained with long term feeding while protein amount decreased, suggesting influence by mechanisms other than, or in addition to, transcriptional regulation. Thus, response patterns and interactions with length of feeding may differ, depending upon the types of vegetables and enzymes, requiring caution when interpreting the results of short-term feeding studies.

Phenethyl isothiocyanate and indole-3-carbinol from cruciferous vegetables, but not furanocoumarins from apiaceous vegetables, reduced PhIP-induced DNA adducts in Wistar rats.

SCOPE: We previously showed that apiaceous but not cruciferous vegetables reduced DNA adducts formed by 2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine (PhIP) in rats. Here, we report the effects of the putative chemopreventive phytochemicals from these vegetables on PhIP metabolism and DNA adduct formation. METHODS AND RESULTS: Rats received three supplemented diets: P + I (phenethyl isothiocyanate and indole-3-carbinol), furanocoumarins (FC, 5-methoxypsoralen, 8-methoxypsoralen, and isopimpinellin), and combination (P + I and FC). Phytochemical supplementation matched the levels in vegetables fed in our previous study. After 6 days, rats were injected with PhIP (10 mg/kg body wt) and killed after 24-h urine collection. Compared to the control, P + I increased activity of hepatic cytochrome P450 (CYP) 1A1 (10.1-fold), CYP1A2 (3.62-fold), and sulfotransferase 1A1 (2.70-fold). The combination diet also increased CYP1A1 and CYP1A2 activity. Urinary metabolomics revealed that PhIP metabolite profiles generally agreed with biotransformation enzyme activities. P + I and combination diets reduced PhIP-DNA adducts by 43.5 and 24.1%, respectively, whereas FC had no effect on adducts, compared to the control diet. CONCLUSION: Effects of phytochemicals on metabolic outcomes and markers of carcinogenesis might differ from fresh vegetables, thus limiting the inferences that one can draw from the effects of purified phytochemicals on the health benefits of the vegetables from which they derive.

Contamination of deconjugation enzymes derived from Helix pomatia with the plant bioactive compounds 3,3'-diindolylmethane, 5-methoxypsoralen, and 8-methoxypsoralen.

Bioactive compounds from plant foods are intensely investigated for effects on disease prevention. ß-Glucuronidase/arylsulfatase from Helix pomatia (snail) is commonly used when quantifying exposure to metabolized dietary components. However, we describe here the contamination of multiple formulations of this enzyme preparation with 3,3'-diindolylmethane (DIM), 8-methoxypsoralen (8-MOP), and 5-methoxypsoralen (5-MOP), bioactives from cruciferous and apiaceous vegetables under investigation as putative cancer chemopreventive agents. We identified an Escherichia coli preparation of ß-glucuronidase as free from contamination with any of the compounds tested. These results demonstrate the importance of selecting appropriate enzyme preparations when quantifying naturally occurring, trace level compounds in biological fluids.