M.H.M. van Herwijnen

Compound-specific effects of diverse neurodevelopmental toxicants on global gene expression in the neural embryonic stem cell test (ESTn)

Alternative assays for developmental toxicity testing are needed to reduce animal use in regulatory toxicology. The in vitro murine neural embryonic stem cell test (ESTn) was designed as an alternative for neurodevelopmental toxicity testing. The integration of toxicogenomic-based approaches may further increase predictivity as well as provide insight into underlying mechanisms of developmental toxicity. In the present study, we investigated concentration-dependent effects of six mechanistically diverse compounds, acetaldehyde (ACE), carbamazepine (CBZ), flusilazole (FLU), monoethylhexyl phthalate (MEHP), penicillin G (PENG) and phenytoin (PHE), on the transcriptome and neural differentiation in the ESTn. All compounds with the exception of PENG altered ESTn morphology (cytotoxicity and neural differentiation) in a concentration-dependent manner. Compound induced gene expression changes and corresponding enriched gene ontology biological processes (GO-BP) were identified after 24h exposure at equipotent differentiation-inhibiting concentrations of the compounds. Both compound-specific and common gene expression changes were observed between subsets of tested compounds, in terms of significance, magnitude of regulation and functionality. For example, ACE, CBZ and FLU induced robust changes in number of significantly altered genes (≥ 687 genes) as well as a variety of GO-BP, as compared to MEHP, PHE and PENG (≤ 55 genes with no significant changes in GO-BP observed). Genes associated with developmentally related processes (embryonic morphogenesis, neuron differentiation, and Wnt signaling) showed diverse regulation after exposure to ACE, CBZ and FLU. In addition, gene expression and GO-BP enrichment showed concentration dependence, allowing discrimination of non-toxic versus toxic concentrations on the basis of transcriptomics. This information may be used to define adaptive versus toxic responses at the transcriptome level.

Effects of polyunsaturated fatty acids on prostaglandin synthesis and cyclooxygenase-mediated DNA adduct formation by heterocyclic aromatic amines in human adenocarcinoma colon cells

Dietary heterocyclic aromatic amines (HCA) and polyunsaturated fatty acids (PUFA) are both believed to play a role in colon carcinogenesis, and are both substrate for the enzyme cyclooxygenase (COX). In HCA‐7 cells, highly expressing isoform COX‐2, we investigated the effects of PUFA on prostaglandin synthesis and DNA adduct formation by the HCA 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP) and 2‐amino‐3‐methylimidazo[4,5‐f]quinoline (IQ). Furthermore, we studied the role of COX, COX‐2 in particular, and cytochrome P4501A2 (CYP1A2) by using the enzyme inhibitors indomethacin (IM), NS‐398, and phenethyl isothiocyanate (PEITC), respectively. COX‐mediated formation of prostaglandin E2 (PGE2) from linoleic acid (LA) showed that HCA‐7 cells can convert LA into arachidonic acid (AA). Alternatively, eicosapentaenoic acid (EPA) was found to compete with AA for COX. Strongly decreased PGE2 levels by addition of IM demonstrated involvement of COX in PUFA metabolism. Both IM and NS‐398 inhibited adduct formation by HCA to nearly the same extent, indicating involvement of COX‐2 rather than COX‐1, while CYP1A2 activity in HCA‐7 cells was demonstrated by addition of PEITC. Overall, inhibiting effects were stronger for PhIP than for IQ. HCA‐DNA adduct formation was stimulated by addition of PUFA, although high PUFA concentrations partly reduced this stimulating effect. Finally, similar effects for n‐3 and n‐6 fatty acids suggested that adduct formation may not be the crucial mechanism behind the differential effects of PUFA on colon carcinogenesis that have been described. These results show that COX, and COX‐2 in particular, can play a substantial role in HCA activation, especially in extrahepatic tissues like the colon. Furthermore, the obvious interactions between PUFA and HCA in COX‐2 expressing cancer cells may be important in modulating colorectal cancer risk.

Nicotinic acid supplementation: effects on niacin status, cytogenetic damage, and poly(ADP-ribosylation) in lymphocytes of smokers.

As a substrate for poly(ADP-ribose) polymerase (PARP; EC, 2.4.2.30), an enzyme that is activated by DNA strand breaks and is thought to facilitate efficient DNA repair, NAD+ and its precursor nicotinic acid (niacin) are involved in the cellular defense against DNA damage by genotoxic compounds. In this study, the effect of nicotinic acid supplementation on cytogenetic damage and poly(ADP-ribosylation) was evaluated in a human population that is continuously exposed to genotoxic agents, e.g., smokers. By use of a placebo-controlled intervention design, 21 healthy smokers received supplementary nicotinic acid at 0-100 mg/day for 14 weeks. An increased niacin status, as assessed from blood nicotinamide concentrations and lymphocyte NAD+ concentrations, was observed in groups supplemented with 50 and 100 mg/day. This effect was most pronounced in subjects with lower initial NAD+ levels. An increased niacin status did not result in decreased hypoxanthine guanine phosphoribosyltransferase variant frequencies and micronuclei induction in peripheral blood lymphocytes (PBLs). Sister chromatid exchanges in PBLs, however, were increased after supplementation with nicotinic acid. This increase was positively associated with the daily dose of nicotinic acid. No effects of nicotinic acid supplementation were found for ex vivo (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene-induced poly(ADP-ribosylation), although the small number of samples that could be analyzed (n = 12) does not allow firm conclusions. Because no evidence was found for a decrease in cigarette smoke-induced cytogenetic damage in PBLs of smokers after nicotinic acid supplementation of up to 100 mg/day, it is concluded that supplemental niacin does not contribute to a reduced genetic risk in healthy smokers.

Benzo[a]pyrene-induced transcriptomic responses in primary hepatocytes and in vivo liver: Toxicokinetics is essential for in vivo–in vitro comparisons

The traditional 2-year cancer bioassay needs replacement by more cost-effective and predictive tests. The use of toxicogenomics in an in vitro system may provide a more high-throughput method to investigate early alterations induced by carcinogens. Recently, the differential gene expression response in wild-type and cancer-prone Xpa−/−p53+/− primary mouse hepatocytes after exposure to benzo[a]pyrene (B[a]P) revealed downregulation of cancer-related pathways in Xpa−/−p53+/− hepatocytes only. Here, we investigated pathway regulation upon in vivo B[a]P exposure of wild-type and Xpa−/−p53+/− mice. In vivo transcriptomics analysis revealed a limited gene expression response in mouse livers, but with a significant induction of DNA replication and apoptotic/anti-apoptotic cellular responses in Xpa−/−p53+/− livers only. In order to be able to make a meaningful in vivo–in vitro comparison we estimated internal in vivo B[a]P concentrations using DNA adduct levels and physiologically based kinetic modeling. Based on these results, the in vitro concentration that corresponded best with the internal in vivo dose was chosen. Comparison of in vivo and in vitro data demonstrated similarities in transcriptomics response: xenobiotic metabolism, lipid metabolism and oxidative stress. However, we were unable to detect cancer-related pathways in either wild-type or Xpa−/−p53+/− exposed livers, which were previously found to be induced by B[a]P in Xpa−/−p53+/− primary hepatocytes. In conclusion, we showed parallels in gene expression responses between livers and primary hepatocytes upon exposure to equivalent concentrations of B[a]P. Furthermore, we recommend considering toxicokinetics when modeling a complex in vivo endpoint with in vitro models.

Biomonitoring the intake of garlic via urinary excretion of allyl mercapturic acid

Allium vegetables (onions, leeks, chives) and in particular garlic have been claimed to have health-promoting potential. This study was conducted to get insight into the perspectives for monitoring the intake of garlic by a biomarker approach. Chemically, the biomarker results from exposure to gamma-glutamyl-S-allyl-l-cysteine, which is first hydrolysed by gamma-glutamine-transpeptidase resulting in the formation of S-allyl-l-cysteine. The latter compound is subsequently N-acetylated by N-acetyltransferase into S-allyl-mercapturic acid (ALMA) and excreted into urine. The mercapturic acid was measured in urine using gaschromatography with mass spectrometry. Thus the intake of garlic was determined to check the compliance of garlic intake in a placebo-controlled intervention study. Results indicate that S-allyl-mercapturic acid could be detected in 15 out of 16 urine samples of garlic supplement takers, indicating good compliance. In addition, the intake of garlic was also monitored in a cross-section study of vegans versus controls in Finland, in which no differences in garlic consumption nor in ALMA output were recorded between vegans and controls. These data indicate good possibilities for further studies in the field of biomarkers to investigate the putative chemopreventive effects of garlic and garlic-containing products.