Identification of biomarkers to detect residual pertussis toxin using microarray analysis of dendritic cells.

In this study we aimed to identify genes that are responsive to pertussis toxin (PTx) and might eventually be used as biological markers in a testing strategy to detect residual PTx in vaccines. By microarray analysis we screened six human cell types (bronchial epithelial cell line BEAS-2B, fetal lung fibroblast cell line MRC-5, primary cardiac microvascular endothelial cells, primary pulmonary artery smooth muscle cells, hybrid cell line EA.Hy926 of umbilical vein endothelial cells and epithelial cell line A549 and immature monocyte-derived dendritic cells) for differential gene expression induced by PTx. Immature monocyte-derived dendritic cells (iMoDCs) were the only cells in which PTx induced significant differential expression of genes. Results were confirmed using different donors and further extended by showing specificity for PTx in comparison to Escherichia coli lipopolysaccharide (LPS) and Bordetella pertussis lipo-oligosaccharide (LOS). Statistical analysis indicated 6 genes, namely IFNG, IL2, XCL1, CD69, CSF2 and CXCL10, as significantly upregulated by PTx which was also demonstrated at the protein level for genes encoding secreted proteins. IL-2 and IFN-γ gave the strongest response. The minimal PTx concentrations that induced production of IL-2 and IFN-γ in iMoDCs were 12.5 and 25IU/ml, respectively. High concentrations of LPS slightly induced IFN-γ but not IL-2, while LOS and detoxified pertussis toxin did not induce production of either cytokine. In conclusion, using microarray analysis we evaluated six human cell lines/types for their responsiveness to PTx and found 6 PTx-responsive genes in iMoDCs of which IL2 is the most promising candidate to be used as a biomarker for the detection of residual PTx.

Formation and removal of genotoxic activity during UV/H(2)O(2)-GAC treatment of drinking water.

The objective of this study was to determine the genotoxic activity of water after UV/H(2)O(2) oxidation and GAC filtration. Pre-treated surface water from three locations was treated with UV/H(2)O(2) with medium pressure (MP) lamps and passed through granulated activated carbon (GAC). Samples taken before and after each treatment step were extracted and concentrated by solid phase extraction (SPE) and analyzed for genotoxicity using the Comet assay with HepG2 cells and the Ames II assay. The Comet assay showed no genotoxic response in any of the samples. In the Ames II, no genotoxic response was obtained with the TAMix (a mix of six strains), but the TA98 strain showed an increase in genotoxic activity after MP-UV/H(2)O(2) for all three locations. GAC post treatment effectively reduced the activities to control levels at two of the three locations and to below the level of the pre-treated water at one site. The results indicate that UV/H(2)O(2) treatment may lead to the formation of genotoxic by-products, which can be removed by subsequent GAC filtration.

Expression profiling of colon cancer cell lines and colon biopsies: towards a screening system for potential cancer-preventive compounds.

Interest in mechanisms of colon cancer prevention by food compounds is strong and research in this area is often performed with cultured colon cancer cells. In order to assess utility for screening of potential cancer-preventive (food) compounds, expression profiles of 14 human cell lines derived from colonic tissue were measured using cDNA microarrays with 4000 genes and compared with expression profiles in biopsies of human colon tumours and normal tissue. Differences and similarities in the gene expression profiles of the cell lines were analysed by clustering and principal component analysis (PCA). Cytoskeleton genes and immune response genes are two functional classes of genes that contributed to the differences between the cell lines. A subset of 72 colon cancer-specific genes was identified by comparing expression profiles in human colon biopsies of tumour tissue and normal tissue. A separation of the cell lines based on the tumour stage of the original adenocarcinoma was observed after PCA of expression data of the subset of colon cancer-specific genes in the cell lines. The results of this study may be useful in the ongoing research into mechanisms of cancer prevention by dietary components.

Do aberrant crypt foci have predictive value for the occurrence of colorectal tumours? Potential of gene expression profiling in tumours.

The effects of different dietary compounds on the formation of aberrant crypt foci (ACF) and colorectal tumours and on the expression of a selection of genes were studied in rats. Azoxymethane-treated male F344 rats were fed either a control diet or a diet containing 10% wheat bran (WB), 0.2% curcumin (CUR), 4% rutin (RUT) or 0.04% benzyl isothiocyanate (BIT) for 8 months. ACF were counted after 7, 15 and 26 weeks. Tumours were scored after 26 weeks and 8 months. We found that the WB and CUR diets inhibited the development of colorectal tumours. In contrast, the RUT and BIT diets rather enhanced (although not statistically significantly) colorectal carcinogenesis. In addition, the various compounds caused different effects on the development of ACF. In most cases the number or size of ACF was not predictive for the ultimate tumour yield. The expression of some tumour-related genes was significantly different in tumours from the control group as compared to tumours from the treated groups. It was concluded that WB and CUR, as opposed to RUT and BIT, protects against colorectal cancer and that ACF are unsuitable as biomarker for colorectal cancer. Effects of the different dietary compounds on metalloproteinase 1 (TIMP-1) expression correlated well with the effects of the dietary compounds on the ultimate tumour yield.

Safety evaluation of an alpha-cyclodextrin glycosyltranferase preparation.

Alpha-cyclodextrin glucosyltransferase (alpha-CGTase, EC 2.4.1.19) is an amylolytic enzyme used for the production of alpha-cyclodextrin (alpha-CD), a novel, soluble dietary fiber, from food-grade starch. The safety of an alpha-CGTase preparation obtained by batch fermentation from a recombinant strain of Escherichia coli K12 harboring the alpha-CGTase gene from Klebsiella oxytoca strain M5a1 was examined. In a 13-week subchronic toxicity study in rats, the administration by gavage of the alpha-CGTase preparation at levels of up to 20 ml/kg bw/day, corresponding to a total organic solids dosage of 260 mg/kg bw/day, did not cause any systemic toxic effect. Some signs of irritation were observed in the respiratory tract which occurred, however, in one sex only and/or were not dose-related. Accordingly, these changes were considered to be an unspecific consequence of the reflux and aspiration of the dosing solution. There was no evidence of a genotoxic activity in Ames tests and a chromosome aberration test in cultured human lymphocytes. It is concluded that the examined alpha-CGTase preparation is safe when used for the production of alpha-CD.

Determination of N-nitrosodimethylamine in artificial gastric juice by gas chromatography-mass spectrometry and by gas chromatography-thermal energy analysis.

The thermal energy analyser (TEA) is considered to be the gold standard for the determination of nitrosamines. However, since many laboratories cannot justify the use of such a very specific detection system, alternative detection methods are useful. While standard gas chromatography (GC) detectors lack the selectivity of the TEA detector, mass spectrometry (MS) seems to be the method of choice to combine GC separation with mass selective detection. Moreover, the detection limits of the GC-MS assay in general use are about 4 times lower than those of the GC-TEA assay. A comparison of GC-MS and GC-TEA data on N-nitrosodimethylamine determinations showed a strong correlation between the two assays (R2 = 0.86), demonstrating the exchangeability of these methods.

Antimutagenic activity of green tea and black tea extracts studied in a dynamic in vitro gastrointestinal model.

An in vitro gastrointestinal model, which simulates the conditions in the human digestive tract, was used to determine potential antimutagenic activity of extracts of black tea and green tea. In this paper, results are presented on the availability for absorption of potential antimutagenic compounds present in tea and on the influence of the food matrix on this activity. Between 60 and 180min after the tea was introduced into the model, antimutagenic activity was recovered from the jejunal compartment by means of dialysis: the dialysate appeared to inhibit the mutagenicity of the food mutagen MeIQx in the direct plate assay with Salmonella typhimurium (Ames test). The maximum inhibition was measured at 2h after the start of the experiment and was comparable for black tea and green tea extract. To determine the influence of food matrices on the antimutagenic activity of tea, the model was loaded with black tea together with milk or a homogenized standard breakfast. The maximum inhibition observed with black tea was reduced by 22, 42 and 78% in the presence of whole milk, semi-skimmed milk, and skimmed milk, respectively. Whole milk and skimmed milk abolished the antimutagenic activity of green tea by more than 90%; for semi-skimmed milk the inhibition was more than 60%. When a homogenized breakfast was added into the model together with the black tea extract, the antimutagenic activity was completely eliminated. When tea and MeIQx were added together into the digestion model, MeIQx mutagenicity was efficiently inhibited, with green tea showing a slightly stronger antimutagenic activity than black tea. In this case, the addition of milk had only a small inhibiting effect on the antimutagenicity. Antioxidant capacity and the concentration of catechins were also measured in the jejunal dialysates. The reduction in antimutagenic activity corresponded with reduction in antioxidant capacity and with a decrease of concentration of three catechins, viz. catechin, epigallocatechin gallate and epigallocatechin. The in vitro gastrointestinal model appears to be a useful tool to study the antimutagenicity of food components.

Application of a dynamic in vitro gastrointestinal tract model to study the availability of food mutagens, using heterocyclic aromatic amines as model compounds.

The TNO gastro-Intestinal tract Model (TIM) is a dynamic computer-controlled in vitro system that mimics the human physiological conditions in the stomach and small intestine. In the current TIM physiological parameters such as pH, temperature, peristaltic movements, secretion of digestion enzymes, bile and pancreatic juices, and absorption of digested products-by removal through dialysis-was simulated. Heterocyclic aromatic amines (HAA; viz. IQ, MeIQ, MeIQx and PhIP) were used as model compounds for food mutagens, and the passage through TIM was investigated for each of these compounds separately. Subsequently, the influence of a matrix and different rates of passage on the availability for absorption and distribution were studied in experiments with prepared meat, supplemented with MeIQx. Samples taken at various time points from the jejunal and ileal dialysates and from the lumen at the end of the small intestine (ileal delivery) were tested for the presence of mutagenic activity in the Ames test with Salmonella typhimurium strain TA98 as indicator, in the presence of mammalian metabolic activation (rat S9 mix). The results show that, comparable with the human in vivo situation, all four HAA are quickly removed (approx. 50% in 2 hr; approx. 95% in 6 hr) and mainly recovered from the lumen into the jejunal and ileal dialysates (94% of recovery). Only 5+/-1.5% is recovered in the chyme at the end of the small intestine. When MeIQx was added to meat, its availability for absorption was slower, although the influence of the gastrointestinal passage time on the availability of MeIQx was more pronounced than this matrix effect. More MeIQx was found in the jejunal dialysate (23%; P<0.01) and less in the ileal delivery (8%; P<0.01) when simulating the gastrointestinal passage of solid meals was compared to simulating that of liquid meals. The present experiments demonstrate that TIM can be applied to study in vitro the availability of heterocyclic aromatic amines in the gastrointestinal tract. More generally, these studies indicate that TIM shows promise as a useful tool for various research purposes dealing with the availability for absorption of mutagenic as well as antimutagenic components in food.

Analysis of urinary caffeine metabolites to assess biotransformation enzyme activities by reversed-phase high-performance liquid chromatography.

An isocratic high-performance liquid chromatography procedure was developed for the analysis of five urinary metabolites of caffeine; caffeine or 1,3,7-trimethylxanthine (137X), paraxanthine or 1,7-dimethylxanthine (17X), 1,7-dimethylurate (17U), 1-methylxanthine (1X), 1-methylurate (1U) and 5-acetylamino-6-formylamino-3-methyluracil (AFMU). A standardized procedure was used for oral intake of caffeine and for urine collection. Conditions for sample storage and preparation were optimized, resulting in no detectable loss of caffeine metabolites after storage of the urine samples for four months. Urine samples were extracted with chloroform-2-propanol (4:1, v/v) and separated on a reversed-phase column with acetic acid (33%)-tetrahydrofuran-acetonitrile-water (1:2.5:44:925.5, v/v) as the eluent. Peaks were monitored at 280 nm. Peak heights were measured and the five metabolites were quantified using calibration curves. Cytochrome P4501A2 (CYP1A2) activity was calculated from the molar ratio (AFMU+1X+1U)/17U, N-acetyltransferase (NAT) from the ratio AFMU/1X, XO from the ratio 1U/1X+1U and cytochrome P4502A6 (CYP2A6) from the ratio 17U/(17U+17X+1U+ 1X+AFMU). The inter-assay coefficients of variation ranged from 1.7% for 17U to 5.7% for IX. The intra-individual variation in metabolite ratios determined in two people, with intervals of a few days to several weeks between measurements, ranged from 2. 1% for XO to 11.0% for CYP2A6. Using this procedure, metabolic ratios were determined for four groups of subjects; healthy, non-smoking females using oral contraceptives (OC users, n=5) and non-users (n=5), healthy nonsmoking males (n=9) and children (n=7). Results found in this study were comparable to results reported in the literature for subjects with similar characteristics. A significantly higher CYP1A2 ratio was found for males (4.87+/-0.47) compared to females (3.62+/-0.91: p=0.005, Mann-Whitney). For the other enzyme activities, no significant differences were found between the groups of subjects in this study.

Influence of nucleotide excision repair on N-hydroxy-2-acetylaminofluorene-induced mutagenesis studied in lambda lacZ-transgenic mice.

To study the influence of nucleotide excision repair (NER) on mutagenesis in vivo, ERCC1 +/-, XPA-/-, and wild-type (ERCC1+/+ and XPA+/+, respectively) lambda lacZ-transgenic mice were treated i.p. with N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and lacZ mutant frequencies were determined in liver. No significant effect of the treatment on the mutant frequency in wild-type or ERCC1-heterozygous mice was observed. The liver mutant frequency appeared to be significantly increased in treated XPA-/- mice only. To distinguish N-OH-AAF-induced from spontaneous mutations, lacZ mutants derived from treated XPA-/- mice were subjected to DNA-sequence analysis and the spectrum obtained was compared to that established for lacZ mutants in liver of PBS-treated lambda lacZ-transgenic mice of the parent strain 40.6. The N-OH-AAF-induced mutation spectrum appeared to be significantly different from the spontaneous mutation spectrum: the former consisted of mainly (19/22) single bp substitutions targeted at G, of which the majority (12/19) were G:C-->T:A transversions, suggesting that N-(deoxyguanosin-8-yl)-2-aminofluorene [dG-C8-AF], the major DNA adduct in N-OH-AAF-treated mice, is the premutagenic lesion. After analysis of 21 spontaneous mutants, only ten single bp substitutions targeted at G were found, of which five were G:C-->T:A transversions. This study with XPA-/- lambda lacZ-transgenic mice shows that one of the components of NER, that is, the XPA protein, suppresses mutagenesis in vivo.