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In studies of hominin adaptations to fire use, the role of the aryl hydrocarbon receptor (AHR) in the evolution of detoxification has been highlighted, including statements that the modern human AHR confers a significantly better capacity to deal with toxic smoke components than the Neanderthal AHR. To evaluate this, we compared the AHR-controlled induction of cytochrome P4501A1 (CYP1A1) mRNA in HeLa human cervix epithelial adenocarcinoma cells transfected with an Altai-Neanderthal or a modern human reference AHR expression construct, and exposed to 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD). We compared the complete AHR mRNA sequences including the untranslated regions (UTRs), maintaining the original codon usage. We observe no significant difference in CYP1A1 induction by TCDD between Neanderthal and modern human AHR, whereas a 150–1,000 times difference was previously reported in a study of the AHR coding region optimized for mammalian codon usage and expressed in rat cells. Our study exemplifies that expression in a homologous cellular background is of major importance to determine (ancient) protein activity. The Neanderthal and modern human dose–response curves almost coincide, except for a slightly higher extrapolated maximum for the Neanderthal AHR, possibly caused by a 5′-UTR G-variant known from modern humans (rs7796976). Our results are strongly at odds with a major role of the modern human AHR in the evolution of hominin detoxification of smoke components and consistent with our previous study based on 18 relevant genes in addition to AHR , which concluded that efficient detoxification alleles are more dominant in ancient hominins, chimpanzees, and gorillas than in modern humans. [ABSTRACT FROM AUTHOR]

A simple and rapid luminometric assay for the detection of chemical inhibitors of human thyroid peroxidase (hTPO) activity was developed and validated with 10 model compounds. hTPO was derived from the human thyroid follicular cell line Nthy-ori 3-1 and its activity was quantified by measuring the oxidation of luminol in the presence of hydrogen peroxide (H2O2), which results in emission of light at 428 nm. In this assay, hTPO activity was shown to be inhibited by 5 known TPO inhibitors and not inhibited by 5 non-inhibitors. Similar results were obtained with porcine TPO (pTPO). The inhibition of hTPO by the model compounds was also tested with guaiacol and Amplilu Red as alternative indicator substrates. While all substrates allowed the detection of pTPO activity and its inhibition, only the Amplilu Red and luminol-based methods were sensitive enough to allow the quantification of hTPO activity from Nthy-ori 3-1 cell lysates. Moreover, luminol gave results with a narrower 95% confidence interval and therefore more reliable data. Whole extracts of fast-growing Nthy-ori 3-1 cells circumvent the need for animal-derived thyroid organs, thereby reducing costs, eliminating potential contamination and providing the possibility to study human instead of porcine TPO. Overall, the application of luminol and Nthy-ori 3-1 cell lysate for the detection of the disruption of hTPO activity was found to represent a valuable in vitro alternative and a possible candidate for inclusion within a high throughput integrated testing strategy for the detection of compounds that potentially interfere with normal thyroid function in vivo. [ABSTRACT FROM AUTHOR]

ABSTRACT In order to define an in vitro integrated testing strategy (ITS) for estrogenicity, a set of 23 reference compounds representing diverse chemical classes were tested in a series of in vitro assays including proliferation and reporter gene assays. Outcomes of these assays were combined with published results for estrogen receptor (ER) binding assays and the OECD validated BG1Luc ER transcriptional activation (TA) assay and compared with the outcomes of the in vivo uterotrophic assay to investigate which assays most accurately predict the in vivo uterotrophic effect and to identify discrepancies between the in vitro assays and the in vivo uterotrophic assay. All in vitro assays used revealed a reasonable to good correlation ( R2 = 0.62-0.87) with the in vivo uterotrophic assay but the combination of the yeast estrogen bioassay with the U2OS ERα-CALUX assay seems most promising for an ITS for in vitro estrogenicity testing. The main outliers identified when correlating data from the different in vitro assays and the in vivo uterotrophic assay were 4-hydroxytamoxifen, testosterone and to a lesser extent apigenin, tamoxifen and kepone. Based on the modes of action possibly underlying these discrepancies it becomes evident that to further improve the ITS and ultimately replace animal testing for (anti-)estrogenic effects, the selected bioassays have to be combined with other types of in vitro assays, including for instance in vitro models for digestion, bioavailability and metabolism of the compounds under investigation. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Zebrafish embryos were exposed to concentration ranges of selected thyroid-active model compounds in order to assess the applicability of zebrafish-based developmental scoring systems within an alternative testing strategy to detect the developmental toxicity of thyroid-active compounds. Model compounds tested included triiodothyronine (T3), propylthiouracil (PTU), methimazole (MMI), sodium perchlorate (NaClO4) and amiodarone hydrochloride (AMI), selected to represent different modes of action affecting thyroid activity. Tested time windows included 48-120 hours post fertilization (hpf), 0-72 hpf and 0-120 hpf. All tested compounds resulted in developmental changes, with T3 being the most potent. The developmental parameters affected included reflective iridophores, beat and glide swimming, inflated swim bladders, as well as resorbed yolk sacs. These effects are only evident by 120 hpf and therefore an existing General Morphology Score (GMS) system was extended to create a General Developmental Score (GDS) that extends beyond the 72 hpf scoring limit of GMS and includes additional parameters that are affected by exposure to model thyroid-active compounds. Moreover, the GDS is cumulative as it includes not only the scoring of developmental morphologies but also integrates developmental dysmorphologies. Exposures from 48-120 hpf did not provide additional information to exposures from 0-120 hpf. The results indicate that the zebrafish GDS can detect the developmental toxicity of thyroid toxicants and may be of use in an integrated testing strategy to reduce, refine and, in certain cases, replace animal testing. [ABSTRACT FROM AUTHOR]

This study investigates the in vitro effect of eleven thyroid-active compounds known to affect pituitary and/or thyroid weights in vivo, using the proliferation of GH3 rat pituitary cells in the so-called “T-screen,” and of FRTL-5 rat thyroid cells in a newly developed test denoted “TSH-screen” to gain insight into the relative value of these in vitro proliferation tests for an integrated testing strategy (ITS) for thyroid activity. Pituitary cell proliferation in the T-screen was stimulated by three out of eleven tested compounds, namely thyrotropin releasing hormone (TRH), triiodothyronine (T3) and thyroxine (T4). Of these three compounds, only T4 causes an increase in relative pituitary weight, and thus T4 was the only compound for which the effect in the in vitro assay correlated with a reported in vivo effect. As to the newly developed TSH-screen, two compounds had an effect, namely, thyroid-stimulating hormone (TSH) induced and T4 antagonized FRTL-5 cell proliferation. These effects correlated with in vivo changes induced by these compounds on thyroid weight. Altogether, the results indicate that most of the selected compounds affect pituitary and thyroid weights by modes of action different from a direct thyroid hormone receptor (THR) or TSH receptor (TSHR)-mediated effect, and point to the need for additional in vitro tests for an ITS. Additional analysis of the T-screen revealed a positive correlation between the THR-mediated effects of the tested compounds in vitro and their effects on relative heart weight in vivo, suggesting that the T-screen may directly predict this THR-mediated in vivo adverse effect. [ABSTRACT FROM AUTHOR]

Background Many different mechanisms are involved in nutrient-related prevention of colon cancer. In this study, a comprehensive assessment of the spectrum of possible biological actions of the bioactive compound quercetin is made using multiple gene expression analysis. Quercetin is a flavonoid that can inhibit proliferation of tumor cells and reduce the number of aberrant crypt foci, although increase of number of colon tumors was also reported. Aim of the study In order to elucidate possible mechanisms involved in its mode of action the effect of quercetin on expression of 4000 human genes in Caco-2 cells was studied and related to functional effects. Methods Caco-2 cells were exposed to 5 or 50 μM quercetin for 48 hours, differential expression of 4000 human genes was studied using microarrays and related to functional effects. Differentially expressed genes were categorized in seven functional groups: cell cycle and differentiation, apoptosis, tumor suppressor genes and oncogenes, cell adhesion and cell-cell interaction, transcription, signal transduction and energy metabolism. Also, cell proliferation and cell cycle distribution were measured. Results Quercetin (5 μM) downregulated expression of cell cycle genes (for example CDC6, CDK4 and cyclin Dl), downregulated cell proliferation and induced cell cycle arrest in Caco-2 cells. After exposure to 50 μM quercetin cell proliferation decreased to 51.3% of control, and further decrease of the percentage of cells in the G1 phase coincided with an increase of the percentage of cells in the sub-G1 phase. Quercetin upregulated expression of several tumor suppressor genes. In addition, genes involved in signal transduction pathways like beta catenin/ TCF signalling and MAPK signal transduction were influenced by quercetin. Conclusions This study shows that large-scale gene expression analysis in combination with functional assays yields a considerable amount of information on (anti-) carcinogenic potential of food components like quercetin. [ABSTRACT FROM AUTHOR]

Background: Curcumin is a spice and a coloring food compound with a promising role in colon cancer prevention. Curcumin protects against development of colon tumors in rats treated with a colon carcinogen, in colon cancer cells curcumin can inhibit cell proliferation and induce apoptosis, it is an anti-oxidant and it can act as an anti-inflammatory agent. The aim of this study was to elucidate mechanisms and effect of curcumin in colon cancer cells using gene expression profiling. Methods: Gene expression changes in response to curcumin exposure were studied in two human colon cancer cell lines, using cDNA microarrays with four thousand human genes. HT29 cells were exposed to two different concentrations of curcumin and gene expression changes were followed in time (3, 6, 12, 24 and 48 hours). Gene expression changes after short-term exposure (3 or 6 hours) to curcumin were also studied in a second cell type, Caco-2 cells. Results: Gene expression changes (>1.5-fold) were found at all time points. HT29 cells were more sensitive to curcumin than Caco-2 cells. Early response genes were involved in cell cycle, signal transduction, DNA repair, gene transcription, cell adhesion and xenobiotic metabolism. In HT29 cells curcumin modulated a number of cell cycle genes of which several have a role in transition through the G2/M phase. This corresponded to a cell cycle arrest in the G2/M phase as was observed by flow cytometry. Functional groups with a similar expression profile included genes involved in phase-II metabolism that were induced by curcumin after 12 and 24 hours. Expression of some cytochrome P450 genes was downregulated by curcumin in HT29 and Caco-2 cells. In addition, curcumin affected expression of metallothionein genes, tubulin genes, p53 and other genes involved in colon carcinogenesis. Conclusions: This study has extended knowledge on pathways or processes already reported to be affected by curcumin (cell cycle arrest, phase-II genes). Moreover, potential new leads to genes and pathways that could play a role in colon cancer prevention by curcumin were identified. [ABSTRACT FROM AUTHOR]

In the current study, a set of 12 reference compounds was tested in a low-density DNA microchip that contains probes for 11 different estrogen-responsive marker genes. Our results show that the seven most informative marker genes on the chip resulted in fingerprints that correctly predicted the (anti-)estrogenic activity of the model compounds except that of the negative control testosterone. Two marker genes, myeloid leukemia factor-1 interacting protein and ubiquitin-conjugating enzyme E2C, were even capable of correctly predicting the estrogenic potency of all five estrogen receptor (ER) agonists tested and correlated well with the potencies as determined in the MCF-7/BOS proliferation assay and the in vivo uterotrophic assay. In addition, it was demonstrated that the estrogenic responses of testosterone, both in the array tube assay and in the proliferation assay, were partially due to the conversion of testosterone into 17β-estradiol by aromatase but also due to formation of other estrogenic metabolites, the presence and estrogenic potency of which were confirmed by gas chromatography-tandem mass spectrometry analysis and a yeast-based reporter gene assay, respectively. It is concluded that low-density DNA microchip-based fingerprinting in MCF-7/BOS cells for estrogenicity marker genes provides a faster in vitro alternative to the current MCF-7/BOS cell proliferation assay (E-screen)., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The market for food products with additional health benefits is increasing rapidly and tools for identification of bio-functional characteristics of food items are essential. To facilitate the detection of beneficial effects of tomato on gene expression, methods to prepare tomato extracts suitable to test in the EpRE LUX assay and other cell-based reporter gene assays for health-related bioactivity mechanisms, were developed. An isoprenoid-containing chloroform extract of tomato fruit and most individual isoprenoids did not induce electrophile-responsive element (EpRE)-mediated gene expression. A semi-polar extract of tomato fruits, enzymatically hydrolysed to remove the glycosyl residues from the phenolic ingredients was able to induce EpRE-mediated luciferase expression at both mRNA and protein level, which might be partly due to the presence of quercetin, kaempferol, naringenin and naringenin chalcone. It was concluded that induction of EpRE-regulated genes, such as detoxifying phase II and antioxidant enzymes, may contribute to the beneficial health effects of tomato., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A typical antioxidant, N-acetyl-L-cysteine (NAC) generally protects cells from oxidative damage induced by reactive oxygen species (ROS). 9,10-Phenanthrenequinone (9,10-PQ), a major quinone in diesel exhaust particles, produces ROS in redox cycling following two-electron reduction by NAD(P)H:quinone oxidoreductase 1 (NQO1), which has been considered as a cause of its cyto- and genotoxicity. In this study, we show that NAC unexpectedly augments the toxicity of 9,10-PQ in cells with low NQO1 activity. In four human skin cell lines, the expression and the activity of NQO1 were lower than in human adenocarcinoma cell lines, A549 and MCF7. In the skin cells, the cytotoxicity of 9,10-PQ was significantly enhanced by addition of NAC. The formation of DNA double strand breaks accompanying phosphorylation of histone H2AX, was also remarkably augmented. On the other hand, the cyto- and genotoxicity were suppressed by addition of NAC in the adenocarcinoma cells. Two contrasting experiments: overexpression of NQO1 in CHO-K1 cells which originally expressed low NQO1 levels, and knock-down of NQO1 in the adenocarcinoma cell line A549 by transfection of RNAi, also showed that NAC suppressed 9,10-PQ-induced toxicity in cell lines expressing high NQO1 activity and enhanced it in cell lines with low NQO1 activity. The results suggested that dual effects of NAC on the cyto- and genotoxicity of 9,10-PQ were dependent on tissue-specific NQO1 activity., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Proliferation assays based on human cell lines are the most used in vitro tests to determine estrogenic properties of compounds. Our objective was to characterise to what extent these in vitro tests provide alternatives for the in vivo Allen and Doisy test, a uterotrophic assay in immature or ovariectomised rodents with uterus weight as a crucial read-out parameter. In the present study four different human cell lines derived from three different female estrogen-sensitive tissues, i.e. breast (MCF-7/BOS and T47D), endometrial (ECC-1) and ovarian (BG-1) cells, were characterised by investigating their relative ERα and ERβ amounts, as the ERα/ERβ ratio is a dominant factor determining their estrogen-dependent proliferative responses. All four cell lines clearly expressed the ERα type and a very low but detectable amount of ERβ on both the mRNA and protein level, with the T47D cell line expressing the highest level of the ERβ type. Subsequently, a set of reference compounds representing different modes of estrogen action and estrogenic potency were used to investigate the proliferative response in the four cell lines, to determine which cell line most accurately predicts the effect observed in vivo. All four cell lines revealed a reasonable to good correlation with the in vivo uterotrophic effect, with the correlation being highest for the MCF-7/BOS cell line (R²=0.85). The main differences between the in vivo uterotrophic assay and the in vitro proliferation assays were observed for tamoxifen and testosterone. The proliferative response of the MCF-7/BOS cells to testosterone was partially caused by its conversion to estradiol by aromatase or via androstenedione to estrone. It is concluded that of the four cell lines tested, the best assay to include in an integrated testing strategy for replacement of the in vivo uterotrophic assay is the human MCF-7/BOS breast cancer cell line., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Activation of peroxisome proliferator-activated receptor γ (PPARγ) by ligands is associated with beneficial health effects, including anti-inflammatory and insulin-sensitizing effects. The aim of the current study was to develop luciferase reporter gene assays to enable fast and low-cost measurement of PPARγ agonist and antagonist activity. Two reporter gene assays, PPARγ1 CALUX and PPARγ2 CALUX, were developed by stable transfection of U2OS cells with an expression vector for PPARγ1 or PPARγ2 and a pGL3-3xPPRE-tata-luc or pGL4-3xPPRE-tata-luc reporter construct, respectively. PPARγ1 CALUX and PPARγ2 CALUX cells showed similar concentration-dependent luciferase induction upon exposure to the PPARγ agonists rosiglitazone, troglitazone, pioglitazone, ciglitazone, netoglitazone, and 15-deoxy-Δ(12,14)-prostaglandin J(2). The potency to induce luciferase decreased in the following order: rosiglitazone>troglitazone=pioglitazone>netoglitazone>ciglitazone. A concentration-dependent decrease in the response to 50nM rosiglitazone was observed on the addition of PPARγ antagonist GW9662 or T0070907 in both PPARγ1 CALUX and PPARγ2 CALUX cells. The PPARα agonists WY14643 and fenofibrate failed to induce luciferase activity, confirming the specificity of these cell lines for PPARγ agonists. In conclusion, PPARγ1 CALUX and PPARγ2 CALUX cells provide a reliable and useful tool to screen (bio)chemicals for PPARγ agonist or antagonist activity., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Cafestol and kahweol are diterpene compounds present in unfiltered coffees. Cafestol is known as the most potent cholesterol-raising agent that may be present in the human diet. Remarkably, the mechanisms behind this effect have only been partly resolved so far. Even less is known about the metabolic fate of cafestol and kahweol. From the structure of cafestol, carrying a furan moiety, we hypothesized that epoxidation may not only be an important biotransformation route but that this also plays a role in its effects found. In bile duct-cannulated mice, dosed with cafestol, we were able to demonstrate the presence of epoxy-glutathione (GSH) conjugates, GSH conjugates and glucuronide conjugates. In addition, it was shown that cafestol was able to induce an electrophile-responsive element (EpRE). Using a murine hepatoma cell line with a luciferase reporter gene under control of an EpRE from the human NQO1 regulatory region, we also found that metabolic activation by CYP450 enzymes is needed for EpRE induction. Furthermore, raising intracellular GSH resulted in a decrease in EpRE-mediated gene induction, whereas lowering intracellular GSH levels increased EpRE-mediated gene induction. In conclusion, evidence suggests that cafestol induces EpRE, apparently via a bioactivation process that possibly involves epoxidation of the furan ring. The epoxides themselves appear subject to conjugation with GSH. The effects on EpRE can also explain the induction of GSH which seems to be involved in the reported beneficial effects of cafestol, for example, when administered with aflatoxin B1 or other toxic or carcinogenic compounds., (Copyright 2010 Elsevier Inc. All rights reserved.)

Isothiocyanates are electrophiles that are able to induce phase II biotransformation enzyme gene expression via an electrophile-responsive element (EpRE) in the gene regulatory region. To study the potency of different isothiocyanates to induce the expression of EpRE-regulated genes, a Hepa-1c1c7 luciferase reporter cell line was exposed to structurally different isothiocyanates. The reporter cell line, EpRE(mGST-Ya)-LUX, contains the EpRE from the regulatory region of the mouse glutathione S-transferase Ya gene. Isothiocyanates containing a methyl-sulfur side chain, e.g. sulforaphane, showed a lower EC(50) (0.8-3.2 microM) and a comparable induction factor (17-22.4) compared to the structurally different isothiocyanates containing an alkyl or aromatic side chain, e.g. allyl and phenylethyl isothiocyanate (EC(50) 3.9-6.5 microM, induction factor 17.5-23). After 24h of exposure, on average (+/-SD) 23+/-5% of the isothiocyanate was found in the cells and 77% in the cell medium. Isothiocyanates prove to be strong inducers of electrophile-responsive element-mediated gene expression at physiological concentrations. The here described luciferase reporter cell line is a suitable assay to measure the potency of compounds to induce EpRE-regulated gene expression.

Binding and activation of the aryl hydrocarbon receptor (AhR) is thought to be an essential step in the toxicity of the environmental pollutants dioxins and dioxin-like PCBs. However, also a number of natural compounds, referred to as NAhRAs (natural Ah-receptor agonists), which are present in, for example, fruits and vegetables, can bind and activate this receptor. To study their potential effects in humans, we first investigated the effect of the prototypical AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on gene expression in ex vivo exposed freshly isolated human lymphocytes, and compared the resulting gene expression profile with those caused by the well-known NAhRA indolo[3,2-b]carbazole (ICZ), originating from cruciferous vegetables, and by a hexane extract of NAhRA-containing grapefruit juice (GJE). Only ICZ induced a gene expression profile similar to TCDD in the lymphocytes, and both significantly up-regulated CYP1B1 and TIPARP (TCDD-inducible poly (ADP-ribose) polymerase) mRNA. Next, we performed a human intervention study with NAhRA-containing cruciferous vegetables and grapefruit juice. The expression of the prototypical AhR-responsive genes CYP1A1, CYP1B1 and NQO1 in whole blood cells and in freshly isolated lymphocytes was not significantly affected. Also enzyme activities of CYP1A2, CYP2A6, N-acetyltransferase 2 (NAT2) and xanthine oxidase (XO), as judged by caffeine metabolites in urine, were unaffected, except for a small down-regulation of NAT2 activity by grapefruit juice. Examination of blood plasma with DR CALUX showed a 12% increased AhR agonist activity 3 and 24 h after consumption of cruciferous vegetables, but did not show a significant effect of grapefruit juice consumption. We conclude that intake of NAhRAs from food may result in minor AhR-related effects measurable in human blood and urine.

Flavonoids are important bioactive compounds, omnipresent in the human diet, and are reported to be bifunctional inducers. These phytochemicals are able to induce xenobiotic-responsive element (XRE)- and electrophile-responsive element (EpRE)-mediated gene expression, resulting in the induction of biotransformation enzymes. To test whether flavonoid-induced EpRE-mediated gene expression could be the result of upstream XRE-mediated gene expression, several flavonoids were tested for their ability to induce XRE- and EpRE-mediated gene expression using two stably transfected reporter gene cell lines constructed in the same mouse Hepa-1c1c7 hepatoma background. Although classified as bifunctional inducers, all flavonoids were found to induce EpRE- and XRE-mediated gene expression in a different concentration range, which presents an issue not considered by the current definition of a bifunctional inducer. At physiological relevant concentrations, the induction of gene expression via the EpRE transcriptional enhancer element is dominant, leading in particular to elevated levels of EpRE-regulated detoxifying enzymes. Furthermore, these results strongly suggest that EpRE-mediated gene expression induced by flavonoids is not a downstream reaction of XRE-mediated gene expression.

Quercetin is a flavonoid reported to have health-promoting properties. Due to its extensive metabolism to glucuronides in vivo, questions were raised if studies conducted with quercetin aglycone, stating its health-promoting activity, are of actual relevance. Here we show that glucuronides of quercetin, and its methylated forms isorhamnetin and tamarixetin, can induce EpRE-mediated gene expression up to 5-fold. Furthermore, evidence is presented that EpRE-mediated gene induction by these glucuronides involves their deglucuronidation. This indicates that although quercetin-derived glucuronides are the major metabolites present in the systemic circulation, deglucuronidated derivatives are the active compounds responsible for their beneficial EpRE-mediated gene expression effects.

Both environment and genetics contribute to the pathogenesis and prevention of colorectal neoplasia., Nad(p)h: quinone oxidoreductase (NQO1) is a detoxification enzyme that is polymorphic and inducible. We investigated interactions between lifestyle factors and polymorphisms in NQO1 and its key regulatory transcription factor NFE2L2 in colorectal adenoma risk. The NQO1 c.609C>T and g.-718A>G and NFE2L2 g.-650C>A, g.-684G>A and g.-686A>G polymorphisms were determined among 740 Dutch adenoma cases and 698 endoscopy-based controls. Dietary intake was assessed by food frequency questionnaire, other lifestyle information by questionnaire. The NQO1 609CT genotype was associated with a higher adenoma risk (OR 1.27, 95% CI 1.00-1.62) compared with the 609CC genotype, whereas the 609TT genotype was not (OR 1.03, 95% CI 0.56-1.88). The higher risk with the NQO1 609CT-genotype was seen among smokers (OR 1.96, 95% CI 1.40-2.76), but not among nonsmokers (OR 0.91, 95% CI 0.62-1.35; interaction p = 0.030). Fruit and vegetable consumption did not protect smokers from adenomas and did not interact with the NQO1 609C>T polymorphism or the NFE2L2 polymorphisms. A higher adenoma risk seen with high fruit and vegetable consumption among NQO1 -718GG genotypes was absent among -718GA genotypes (interaction p = 0.071). Gene-gene interactions were observed between the NQO1 609C>T and NFE2L2 -686A>G polymorphisms (interaction p = 0.056) and between the NQO1 -718 G>A and NFE2L2 -650C>A polymorphisms (interaction p = 0.013)., In Conclusion: the NQO1 609CT genotype is associated with increased adenoma risk among smokers, which is not diminished by high fruit and vegetable consumption. The observed gene-gene interactions may point to a role for NFE2L2 polymorphisms in NQO1-related adenoma formation.

NAD(P)H:quinone oxidoreductase (NQO1) is an inducible detoxification enzyme relevant for colorectal cancer biochemoprevention. We evaluated the influence of recent fruit and vegetable (F&V) consumption and polymorphisms in NQO1 and transcription factor NFE2L2 on rectal NQO1 phenotype and also whether white blood cell (WBC) NQO1 activity reflects rectal activity. Among 94 sigmoidoscopy patients, we assessed F&V consumption by dietary record and determined the NQO1 c.609C > T and g.-718A > G and NFE2L2 g.-650C > A, g.-684G > A, and g.-686A > G polymorphisms. NQO1 mRNA level was measured in rectal biopsies and NQO1 activity in rectal biopsies and WBC. Consumption of F&V did not yield higher mRNA level or activity but rather appeared to have a repressive effect. Rectal activity was higher among NQO1 609CC-genotypes as compared to 609CT-genotypes (P < 0.0001; 609TT-genotypes were absent), whereas mRNA was higher among 609CT-genotypes (P < 0.001). mRNA and activity correlated among NQO1 609CC-genotypes (r = .50, P = 0.0001) but not among 609CT-genotypes (r = .14, P = 0.45). The NFE2L2-684A-allele was associated with higher mRNA levels (P = < 0.05). The other polymorphisms did not affect phenotype significantly. WBC and rectal activity did not correlate. In conclusion, genetic variation, especially the NQO1 609C > T polymorphism, is a more important predictor of rectal NQO1 phenotype than F&V consumption. WBC NQO1 activity is not a good surrogate for rectal activity.

High glutathione S-transferase (GST) activity may contribute to colorectal cancer prevention. Functional polymorphisms are known in the GSTM1, GSTT1, GSTA1 and GSTP1 genes. The influence of these GST polymorphisms and recent fruit and vegetable consumption on GST levels and activity has not been investigated simultaneously in a human population. Also, it is not clear if blood GST activity reflects rectal GST activity. Therefore, we determined GST polymorphisms in 94 patients scheduled for sigmoidoscopy. Rectal GST isoenzyme levels (GSTM1, GSTM2, GSTT1, GSTA and GSTP1) were measured by quantitative western blotting, and rectal and white blood cell total GST activities were measured spectrophotometrically using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate. Vegetable and fruit consumption was assessed by dietary record. As expected, the GSTM1 and GSTT1 deletion polymorphisms, and the GSTA1 g.-69C-->T polymorphism significantly affected the respective isoenzyme levels. Also, rectal GST isoenzyme levels differed between those with and without recent consumption of Alliaceae, Cucurbitaceae, Apiaceae and citrus fruit. Rectal GST activity, however, was not clearly influenced by fruit and vegetable consumption. It was most significantly determined by the GSTP1 c.313A-->G polymorphism; compared with the 313AA genotypes, the 313AG and 313GG genotypes showed 36 and 67 nmol/min/mg protein (P < 0.001) lower GST activity, respectively. The correlation between rectal and white blood cell GST activities was low (r = 0.40, P < 0.001), and the relevance of the various genetic and dietary factors appeared to differ between the two tissues. In conclusion, this study indicates that the GST enzyme system is influenced by both GST polymorphisms and consumption of fruits and vegetables. The latter appeared more important for individual rectal GST isoenzyme levels than for total GST activity, which could affect detoxification of isoenzyme-specific substrates. The study results do no support the use of white blood cell GST activity as a surrogate measure for rectal GST activity.

In this study the n-3 polyunsaturated fatty acids (PUFAs) eicosapentaenoic acid and docosahexaenoic acid appear to be effective inducers of electrophile-responsive element (EpRE) regulated genes, whereas the n-6 PUFA arachidonic acid is not. These n-3 PUFAs need to be oxidized to induce EpRE-regulated gene expression, as the antioxidant vitamin E can partially inhibit the PUFA induced dose-dependent effect. Results were obtained using a reporter gene assay, real-time RT-PCR and enzyme activity assays. The induction of EpRE-regulated phase II genes by n-3 PUFAs may be a major pathway by which n-3 PUFAs, in contrast to n-6 PUFAs, are chemopreventive and anticarcinogenic.

NAD(P)H:quinone oxidoreductase (NQO1)-mediated detoxification of quinones is suggested to be involved in cancer prevention. In the present study, using transfected CHO cells, it was demonstrated that the relation between NQO1 activity and the resulting protection against the cytotoxicity of menadione shows a steep dose-response curve revealing a 'lower protection threshold' of 0.5mumol DCPIP/min/mg protein and an 'upper protection threshold' at 1mumol DCPIP/min/mg protein. In an additional in vivo experiment it was investigated how both in vitro critical activity levels of NQO1, relate to NQO1 activities in mice and man, either without or upon induction of the enzyme by butylated hydroxyanisol (BHA) or indole-3-carbinol (I(3)C). Data from an experiment with CD1 mice revealed that base-line NQO1 levels in liver, kidney, small intestine, colon and lung are generally below the observed 'lower protection threshold' in vitro, this also holds for most human tissue S-9 samples. To achieve NQO1 levels above this 'lower protection threshold' will require 5-20 fold NQO1 induction. Discussion focuses on the relevance of the in vitro NQO1 activity thresholds for the in vivo situation. We conclude that increased protection against menadione toxicity can probably not be achieved by NQO1 induction but should be achieved by other mechanisms. Whether this conclusion also holds for other electrophiles and the in vivo situation awaits further definition of their NQO1 protection thresholds.

The electrophile-responsive element (EpRE) is a transcriptional enhancer involved in cancer-chemoprotective gene expression modulation by certain food components. Two stably transfected luciferase reporter cell lines were developed, EpRE(hNQO1)-LUX and EpRE(mGST-Ya)-LUX, based on EpRE sequences from the human NAD(P)H:quinone oxidoreductase (hNQO1) and the mouse glutathione-S-transferase Ya (mGST-Ya) gene, containing one and two tandem EpRE core sequences, respectively. The standard inducer tert-butylhydroquinone (tBHQ), the electrophile benzyl isothiocyanate (BITC), and the antioxidant flavonoid quercetin were found to induce luciferase expression, thereby validating these newly developed reporter cell lines. For tBHQ and BITC, but not for quercetin, higher maximum luciferase induction was found under control of the mGST-Ya EpRE as compared to the hNQO1 EpRE, pointing at different induction mechanisms. Furthermore, we investigated the structure-activity relationship for induction of luciferase expression by flavonoids in EpRE(mGST-Ya)-LUX cells, and also the relation between luciferase induction and flavonoid antioxidant potency. Five different flavonoids with a planar molecular structure were found to induce various levels of luciferase activity, whereas taxifolin, a non-planar flavonoid, did not induce luciferase activity. This suggests that a stereospecific molecular interaction may be important for EpRE-mediated gene activation, possibly with Keap1, a regulator of EpRE-controlled transcription, or with another effector or receptor protein. No consistent relation between luciferase induction level and flavonoid antioxidant potential was observed. Altogether, these results point to differences in induction mechanism between the various chemoprotective compounds tested. The newly developed stably transfected reporter cell lines provide a validated tool for future screening and mechanistic studies of EpRE-mediated gene transcription.

The possible interplay between cruciferous vegetable consumption, functional genetic variations in glutathione S-transferases (GST) M1, T1, P1, and A1, and colorectal adenomas, was investigated in a Dutch case-control study. The GSTM1 and GSTT1 deletion polymorphisms, and the single nucleotide polymorphisms in GSTP1 (A313G) and in GSTA1 (C-69T) were assessed among 746 cases who developed colorectal adenomas and 698 endoscopy-based controls without any type of colorectal polyps. High and low cruciferous vegetable consumption was defined based on a median split in the control group. High consumption was slightly positively associated with colorectal adenomas [odds ratio (OR) 1.15; 95% confidence interval, 0.92-1.44]. For GSTP1, a positive association with higher cruciferous vegetable intake was only apparent in individuals with the low-activity GSTP1 genotype (GG genotype, OR 1.94; 95% confidence interval, 1.02-3.69). This interaction was more pronounced in men, with higher age and with higher meat intake. The GSTA1 polymorphism may have a modifying role as well: the OR for higher intake compared with lower intake was 1.57 (0.93-2.65) for individuals homozygous for the low expression variant (TT genotype). This seemed to be stronger with younger age and higher red meat intake. Cruciferous vegetable consumption and the combined GSTA1 and GSTP1 genotypes showed a statistically significant interaction (P = 0.034). The GSTM1 and GSTT1 genotypes did not seem to modify the association between cruciferous vegetable intake and colorectal adenomas. In conclusion, GSTP1 and GSTA1 genotypes might modulate the association between cruciferous vegetable intake and colorectal adenomas.

Procedures for assessing enzyme inhibition in living cells are an important tool in the study of the relevance of enzyme-catalyzed reactions and interactions in the human body. This paper presents the effects of flavonoids on NAD(P)H:quinone oxidoreductase 1 (NQO1) activity, by a newly developed method to measure NQO1 inhibition in intact cells. The principle of this method is based on the resorufin reductase activity of NQO1. The change in fluorescence in time was used to determine NQO1 activity in intact Chinese hamster ovary (CHO) cells genetically engineered to overexpress human NQO1. Applying this method to determine the inhibitory effects of reported in vitro NQO1 inhibitors (dicoumarol, 7,8-dihydroxyflavone, chrysin) showed that for all inhibitors tested, the IC50 in intact cells was at least 3 orders of magnitude higher than the IC50 in cell lysates. This result demonstrates that in vitro studies with purified NQO1 or with extracts from disrupted tissues are of limited value for obtaining insight into the situation in living cells. Possible factors underlying this discrepancy are being discussed. For the first time, we determined NQO1 inhibition by flavonoids in cells without disruption of the cells or addition of cofactors, enabling the assessment of enzymatic activity and the interaction of modulators of enzymatic activity in an intracellular situation.

This paper describes the screening of 22 extracts from 18 different aquatic environmental samples for androgenic activity, including indirect and interactive effects on androgen receptor (AR)-mediated signal transduction, using the AR-LUX bioassay. Four samples, originating from an industrial wastewater treatment plant (WTP) or the river Meuse, were shown to contain substantial androgenic activity. Moreover, the samples originating from the industrial WTP showed an enhancement of the maximal androgenic response relative to that elicited by the standard androgen methyltrienolone (R1881) in the AR-LUX assay. This indicates the involvement of cellular mechanisms other than receptor-ligand interaction influencing AR-regulated pathways. This also demonstrates the additional value of cell based assays featuring a more complete array of fully functional interacting pathways. Chemical analysis using GC-MS confirmed the presence of a number of androgens and also estrogens in these WTP samples. Subsequently, we showed that estrone and tributyltin hydride (TBT-H) enhance the response to androgens. This indicates that the presence of numerous compounds in addition to androgens in environmental mixtures might very well result in a more profound perturbation of the normal physiology of exposed organisms than estimated based on the androgen levels alone. Therefore, risk assessment of environmental samples should include an evaluation of the presence and the interactive effects of (ant)agonists of carefully selected relevant cellular receptors in order to provide a realistic estimate of the integrated ecotoxicological risk of the compounds present.

The effects of quercetin on viability and proliferation of Chinese Hamster Ovary (CHO) cells and CHO cells overexpressing human quinone reductase (CHO+NQO1) were studied to investigate the involvement of the pro-oxidant quinone chemistry of quercetin. The toxicity of menadione was significantly reduced in CHO+NQO1 cells compared to wild-type CHO cells, validating the NQO1-overexpression in the CHO+NQO1 transfectant. Quercetin inhibited the proliferation of wild-type CHO and CHO+NQO1 cells to a similar extent without affecting cell viability, indicating that NQO1 enrichment of CHO cells did not provide increased protection. On the other hand, inhibition of NQO1 in both types of cells by dicoumarol significantly potentiated the inhibitory effect of quercetin on cell proliferation, revealing the role of NQO1 in cellular protection against quercetin. Altogether, these results can be explained by the hypothesis that both wild-type CHO and CHO+NQO1 cells contain sufficient NQO1 activity for optimal protection against the pro-oxidant effect of quercetin on cell proliferation. The results also point at a cellular NQO1 threshold for optimal protection against quercetin. This NQO1 threshold seems to be in the range of NQO1 activities already present in various tissues.

NAD(P)H:quinone oxidoreductase 1 (NQO1) has often been suggested to be involved in cancer prevention by means of detoxification of electrophilic quinones. In the present study, a series of Chinese hamster ovary (CHO) cell lines expressing various elevated levels of human NQO1 were generated by stable transfection. The level of NQO1 over-expression ranged from 14 to 29 times the NQO1 activity in the wild-type CHO cells. This panel of cell lines, allowed investigation of the protective role of NQO1 in quinone cytotoxicity. It could be demonstrated that menadione toxicity was significantly reduced in all NQO1-transfected CHO clones compared to the wild-type cells, but the clones did not show differences in their level of protection against menadione. This observation pointed at a critical threshold concentration of NQO1 above which a further increase does not provide further protection against quinone cytotoxicity. Additional studies in which the NQO1 activity was inhibited by dicoumarol showed that only dicoumarol concentrations of about five times the EC(50) for NQO1 inhibition were able to reduce NQO1 levels below the apparent threshold, making the cells more sensitive. The level of this threshold was estimated to be in the range of base line NQO1 activities observed in several tissues and species. Thus, the results of the present study indicate that beneficial effects of NQO1 induction by, for example, cruciferous vegetables might be absent or present depending on the NQO1 activity threshold for optimal protection and the basal level of NQO1 expression in the tissue and species of interest.