J.H.M. van Delft

The importance of polymerization and galloylation for the antiproliferative properties of procyanidin-rich natural extracts

Grape (Vitis vinifera) and pine (Pinus pinaster) bark extracts are widely used as nutritional supplements. Procyanidin‐rich fractions from grape and pine bark extract showing different mean degrees of polymerization, percentage of galloylation (percentage of gallate esters) and reactive oxygen species‐scavenging capacity were tested on HT29 human colon cancer cells. We observed that the most efficient fractions in inhibiting cell proliferation, arresting the cell cycle in G2 phase and inducing apoptosis were the grape fractions with the highest percentage of galloylation and mean degree of polymerization. Additionally, the antiproliferative effects of grape fractions were consistent with their oxygen radical‐scavenging capacity and their ability to trigger DNA condensation–fragmentation.

Assessing the Metabolic Competence of Sandwich-Cultured Mouse Primary Hepatocytes

Primary human and rat hepatocyte cultures are well established in vitro systems used in toxicological studies. However, whereas transgenic mouse models provide an opportunity for studying mechanisms of toxicity, mouse primary hepatocyte cultures are less well described. The potential usefulness of a mouse hepatocyte-based in vitro model was assessed in this study by investigating time-dependent competence for xenobiotic metabolism and gene expression profiles. Primary mouse hepatocytes, isolated using two-step collagenase perfusion, were cultured in a collagen sandwich configuration. Gene expression profiles and the activities of various cytochrome P450 (P450) enzymes were determined after 0, 42, and 90 h in culture. Principal component analysis of gene expression profiles shows that replicates per time point are similar. Gene expression levels of most phase I biotransformation enzymes decrease to approximately 69 and 57% of the original levels at 42 and 90 h, respectively, whereas enzyme activities for most of the studied P450s decrease to 59 and 34%. The decrease for phase II gene expression is only to 96 and 92% of the original levels at 42 and 90 h, respectively. Pathway analysis reveals initial effects at the level of proteins, external signaling pathways, and energy production. Later effects are observed for transcription, translation, membranes, and cell cycle-related gene sets. These results indicate that the sandwich-cultured primary mouse hepatocyte system is robust and seems to maintain its metabolic competence better than that of the rat hepatocyte system.

The 10 basic requirements for a scientific paper reporting antioxidant, antimutagenic or anticarcinogenic potential of test substances in in vitro experiments and animal studies in vivo

There is increasing evidence that chemicals/test substances cannot only have adverse effects, but that there are many substances that can (also) have a beneficial effect on health. As this journal regularly publishes papers in this area and has every intention in continuing to do so in the near future, it has become essential that studies reported in this journal reflect an adequate level of scientific scrutiny. Therefore a set of essential characteristics of studies has been defined. These basic requirements are default properties rather than non-negotiables: deviations are possible and useful, provided they can be justified on scientific grounds. The 10 basic requirements for a scientific paper reporting antioxidant, antimutagenic or anticarcinogenic potential of test substances in in vitro experiments and animal studies in vivo concern the following areas: (1) Hypothesis-driven study design; (2) The nature of the test substance; (3) Valid and invalid test systems; (4) The selection of dose levels and gender; (5) Reversal of the effects induced by oxidants, carcinogens and mutagens; (6) Route of administration; (7) Number and validity of test variables; (8) Repeatability and reproducibility; (9) Statistics; and (10) Quality Assurance.

A transcriptomics-based in vitro assay for predicting chemical genotoxicity in vivo

The lack of accurate in vitro assays for predicting in vivo toxicity of chemicals together with new legislations demanding replacement and reduction of animal testing has triggered the development of alternative methods. This study aimed at developing a transcriptomics-based in vitro prediction assay for in vivo genotoxicity. Transcriptomics changes induced in the human liver cell line HepG2 by 34 compounds after treatment for 12, 24, and 48 h were used for the selection of gene-sets that are capable of discriminating between in vivo genotoxins (GTX) and in vivo nongenotoxins (NGTX). By combining transcriptomics with publicly available results for these chemicals from standard in vitro genotoxicity studies, we developed several prediction models. These models were validated by using an additional set of 28 chemicals. The best prediction was achieved after stratification of chemicals according to results from the Ames bacterial gene mutation assay prior to transcriptomics evaluation after 24h of treatment. A total of 33 genes were selected for discriminating GTX from NGTX for Ames-positive chemicals and 22 for Ames-negative chemicals. Overall, this method resulted in 89% accuracy and 91% specificity, thereby clearly outperforming the standard in vitro test battery. Transcription factor network analysis revealed HNF3a, HNF4a, HNF6, androgen receptor, and SP1 as main factors regulating the expression of classifiers for Ames-positive chemicals. Thus, the classical bacterial gene mutation assay in combination with in vitro transcriptomics in HepG2 is proposed as an upgraded in vitro approach for predicting in vivo genotoxicity of chemicals holding a great promise for reducing animal experimentations on genotoxicity.

A pro-inflammatory genotype predisposes to Barrett's esophagus

INTRODUCTION Severity of mucosal inflammation is shown to be associated with Barretts esophagus (BE) development in animals. It has therefore been postulated that a strong pro-inflammatory host response predisposes to BE. AIM To determine the impact of cytokine gene polymorphisms on the development of BE. METHODS The multiplex SNaPshot method was used to determine interleukin (IL)-12B (A+1188C), IL-10 (C-592A, C-819T, A-1082G), IL-8 (A-251T), IL-6 (G-174C) and IL-2 (G-330T) gene polymorphisms in 255 patients with BE and 247 patients with reflux esophagitis (RE). RESULTS The presence of the IL-12B C-allele, which is associated with increased IL-12p70 expression, was more frequently observed in BE than in RE patients [odds ratio (OR) 1.8; 95% confidence interval (CI) 1.2-2.7; P = 0.007). The risk of BE was increased in patients in whom the IL-12B C-allele coincided with a hiatal hernia (OR 2.9; 95% CI 1.32-6.58; P = 0.008). The IL-10(-1082) GG genotype, which is associated with higher IL-10 levels, was also associated with a decreased risk of BE when it was associated with the IL-12B C-allele, indicating IL-10-dependent down-regulation of IL-12p70 expression. A combination of the IL-12B AA genotype and the IL-10 AA or AG genotypes was associated with RE (OR 1.4; 95% CI 1.05-1.85; P = 0.011). CONCLUSION A genetic profile predisposing to a strong pro-inflammatory host response, mediated by IL-12p70 and partially dependent on IL-10, is associated with BE. This risk further increases when this genotype coincides with a hiatal hernia, suggesting that exposure to gastroesophageal reflux in the presence of a pro-inflammatory genetic background is a driving force in the development of BE.

Methodologies for bulky DNA adduct analysis and biomonitoring of environmental and occupational exposures

It is undisputed that DNA adduct formation is one of the key processes in early carcinogenesis. Therefore, analysis of DNA adduct levels may be one of the best tools available to characterize exposure to complex mixtures of genotoxic chemicals as occurring in different environmental and occupational exposure settings. However, from an analytical point of view the detection and quantification of DNA adducts is a challenging enterprise as extremely high sensitivity and selectivity are required. The entire spectrum of chromatographic techniques, including thin-layer chromatography (TLC), gas and liquid chromatography as well as capillary electrophoresis has been used in combination with different detection systems, all with their own specific characteristics. Among the various combinations of techniques, the TLC-(32)P-postlabeling combination appears to meet best with criteria of sensitivity and requirements of minimal amounts of material. Recent developments in the application of capillary electrophoresis in combination with either immunochemical or mass spectrometric detection techniques may offer new and promising approaches, with higher selectivity as compared to TLC-(32)P postlabeling. The applicability of these new techniques in biomonitoring studies aiming at the exposure and risk assessment of low and chronic exposures remains to be determined. In this paper we compare and discuss the advantages and limitations of different techniques used in DNA adduct analysis, with specific emphasis on those adducts formed by the polycyclic aromatic hydrocarbons and heterocyclic aromatic amines.

Toxicogenomic profiles in relation to maternal immunotoxic exposure and immune functionality in newborns.

A crucial period for the development of the immune system occurs in utero. This results in a high fetal vulnerability to immunotoxic exposure, and indeed, immunotoxic effects have been reported, demonstrating negative effects on immune-related health outcomes and immune functionality. Within the NewGeneris cohort BraMat, a subcohort of the Norwegian Mother and Child Cohort Study (MoBa), immunotoxicity was demonstrated for polychlorinated biphenyls and dioxins, showing associations between estimated maternal intake levels and reduced measles vaccination responses in the offspring at the age of 3. The present study aimed to investigate this link at the transcriptomic level within the same BraMat cohort. To this end, whole-genome gene expression in cord blood was investigated and found to be associated with maternal Food Frequency Questionnaires-derived exposure estimates and with vaccination responses in children at 3 years of age. Because the literature reports gender specificity in the innate, humoral, and cell-mediated responses to viral vaccines, separate analysis for males and females was conducted. Separate gene sets for male and female neonates were identified, comprising genes significantly correlating with both 2,3,7,8-tetrachlorodibenzodioxin (TCDD) and polychlorinated biphenyls (PCB) exposure and with measles vaccination response. Noteworthy, genes correlating negatively with exposure in general show positive correlations with antibody levels and vice versa. For both sexes, these included immune-related genes, suggesting immunosuppressive effects of maternal exposure to TCDD and PCB at the transcriptomic level in neonates in relation to measles vaccination response 3 years later.

Comparison of Coumarin-Induced Toxicity between Sandwich-Cultured Primary Rat Hepatocytes and Rats in Vivo: A Toxicogenomics Approach

Sandwich-cultured primary rat hepatocytes are often used as an in vitro model in toxicology and pharmacology. However, loss of liver-specific functions, in particular, the decline of cytochrome P450 (P450) enzyme activity, limits the value of this model for prediction of in vivo toxicity. In this study, we investigated whether a hepatic in vitro system with improved metabolic competence enhances the predictability for coumarin-induced in vivo toxicity by using a toxicogenomics approach. Therefore, primary rat hepatocytes were cultured in sandwich configuration in medium containing a mixture of low concentrations of P450 inducers, phenobarbital, dexamethasone, and β-naphthoflavone. The toxicogenomics approach used enabled comparison of similar mechanistic end-points at the molecular level between in vitro and in vivo conditions, namely, compound-induced changes in multiple genes and signaling pathways. Toxicant-induced cytotoxic effects and gene expression profiles observed in hepatocytes cultured in modified medium and hepatocytes cultured in standard medium (without inducers) were compared with results from a rat in vivo study. Coumarin was used as a model compound because its toxicity depends on bioactivation by P450 enzymes. Metabolism of coumarin toward active metabolites, coumarin-induced cytotoxicity, and gene expression modulation were more pronounced in hepatocytes cultured in modified medium compared with hepatocytes cultured in standard medium. In addition, more genes and biological pathways were similarly affected by coumarin in hepatocytes cultured in modified medium and in vivo. In conclusion, these experiments showed that for coumarin-induced toxicity, sandwich-cultured hepatocytes maintained in modified medium better represent the situation in vivo compared with hepatocytes cultured in standard medium.

Gene expression profiling in primary mouse hepatocytes discriminates true from false-positive genotoxic compounds.

Well-established in vitro methods for testing the genotoxic potency of chemicals--such as the Ames/Salmonella test, the mouse lymphoma assay, the micronucleus test and the chromosomal aberration test--show a high false-positive rate for predicting in vivo genotoxicity and carcinogenicity. Thus, there is a need for more reliable in vitro assays. We investigated whether gene expression profiling in metabolically competent primary mouse hepatocytes is capable of discriminating true genotoxic (GTX) compounds from false-positive genotoxic (FP-GTX) compounds. Sandwich-cultured primary hepatocytes from male C57Bl6 mice were treated for 24 and 48 h with five true GTX and five FP-GTX compounds. Whole genome gene expression modifications were analysed by means of Affymetrix mouse genome 430 2.0 microarrays. Filtered genes were used for hierarchical clustering and class prediction methods. Classifiers were generated by prediction analysis of microarray using a leave-one-compound-out method and selecting the genes that were common to the 10 training sets. For the training compounds, all but one were correctly classified. Validation of the classification model with five new compounds resulted in a 100% correct classification at 24 h and 80% at 48 h. The generated classifiers were mostly involved in metabolic and biosynthetic processes, immune responses and apoptosis. Applying genes whose expression change correlates with γH2AX foci, a measure for DNA damage, did not improve the classification. The present study shows that gene expression profiling in primary mouse hepatocytes is capable of discriminating between true GTX and FP-GTX compounds.

Baseline and genotoxic compound induced gene expression profiles in HepG2 and HepaRG compared to primary human hepatocytes.

Efforts are put into developing toxicogenomics-based toxicity testing methods using in vitro human cell models for improving human risk assessment/replacing animal models. Human in vitro liver models include HepG2, HepaRG and primary human hepatocytes (PHH). Studies on comparability/applicability of these cell types mainly focus on assessing baseline biotransformation capacities/cytochrome P450-inducibility, but compound-induced gene expression profiles are at least as important. Therefore, we compared baseline and aflatoxin B1- and benzo(α)pyrene-induced gene expression profiles in HepG2, HepaRG and PHH (11-13 donors). At baseline, all liver models differ from each other with respect to whole genome gene expression levels. PHH show profound inter-individual differences, and are most similar to HepaRG. After compound exposure, induced gene expression profiles are more similar between cell models, especially for benzo(α)pyrene. Pathways involved in compound metabolism are induced in all 3 models, while others are more pronounced in a specific cell model. Examples are transcriptomic modifications of carbohydrate-related genes (HepaRG) and of receptor-related genes (PHH) after benzo(α)pyrene exposure, and of cell cycle-related genes (HepG2) after aflatoxin B1 exposure. PHH gene expression responses are the most heterogeneous. In conclusion, at base line level PHH are more similar to HepaRG than to HepG2, but for toxicogenomics applications both cell lines perform equally well in comparison to PHH.