AhR-agonistic, anti-androgenic, and anti-estrogenic potencies of 2-isopropylthioxanthone (ITX) as determined by in vitro bioassays and gene expression profiling.

2-Isopropylthioxanthone (2-ITX) has been widely used as a photoinitiator in printing ink of packaging materials. A few years ago, this compound got special attention since it was detected in milk and fruit drinks. Since little is known about possible effects of this compound on human health, studies were initiated to investigate its properties, starting with in vitro studies. Structural similarities between ITX and the AhR-agonists 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and furanocoumarins, prompted us to investigate whether ITX could have the potency to activate the AhR. ITX showed a clear response in the DR CALUX bioassay and also induced EROD activity in H4IIE rat hepatoma cells. Microarray studies in these cells showed a gene expression profile similar to that of TCDD. Moreover, using yeast-based estrogen and androgen bioassays, it was demonstrated that ITX, but not TCDD, has potential anti-estrogenic and anti-androgenic properties. The observed in vitro effects warrant further in vivo studies for confirmation and hazard characterization.

A human intervention study with foods containing natural Ah-receptor agonists does not significantly show AhR-mediated effects as measured in blood cells and urine.

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.

PI3K/AKT, JNK, and ERK pathways are not crucial for the induction of cholesterol biosynthesis gene transcription in intestinal epithelial cells following treatment with the potato glycoalkaloid alpha-chaconine.

We previously reported that exposure of the intestinal epithelial Caco-2 cell line to noncytotoxic concentrations of potato glycoalkaloids resulted in increased expression of cholesterol biosynthesis genes. Genes involved in mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/v-akt murine thymoma viral oncogene homologue (AKT) pathways and their downstream effectors such as Jun, c-Myc, and Fos also were induced. MAPK and PI3K/AKT pathways have been described to regulate the activity of sterol regulatory element binding transcription factors (SREBPs) and consequently the expression of cholesterol biosynthesis genes. In this study, to understand the mechanism of induction of cholesterol biosynthesis upon alpha-chaconine treatment, its effect on SREBP-2 protein levels was investigated. We also examined whether MAPK and PI3K/AKT pathways are required for the observed induction of these genes following exposure of cells to alpha-chaconine. Differentiated Caco-2 cells were pretreated with LY294002 (PI3K inhibitor), PD98059 (MEK1 inhibitor), or SP600125 (JNK inhibitor) or a combination of all inhibitors for 24 h prior to coincubation with 10 microM alpha-chaconine for 6 h. Significant increases in precursor and mature protein levels of SREBP-2 were observed after alpha-chaconine exposure. We also observed that alpha-chaconine treatment resulted in significant phosphorylation of AKT, extracellular signal related protein kinase (ERK), and c-jun N terminal protein kinase (JNK) but not that of p38. In general, the kinase inhibitor experiments revealed that phosphorylation of kinases of PI3K/AKT, ERK, and JNK pathways was not crucial for the induction of expression of cholesterol biosynthesis genes, with the exception of SC5DL. The transcription of this later gene was reduced when all three pathways were inhibited. On the basis of these results, it can be postulated that other mechanisms, which may be independent of the MAPK and PI3K/AKT pathways, including possibly post-translational activation of SREBP-2, may be more pivotal for the induction of cholesterol biosynthesis genes following exposure of intestinal cells to alpha-chaconine.

High folic acid increases cell turnover and lowers differentiation and iron content in human HT29 colon cancer cells.

Folate, a water-soluble B vitamin, is a cofactor in one-carbon metabolism and is essential for DNA synthesis, amino acid interconversion, methylation and, consequently, normal cell growth. In animals with existing pre-neoplastic and neoplastic lesions, folic acid supplementation increases the tumour burden. To identify processes that are affected by increased folic acid levels, we compared HT29 human colon cancer cells exposed to a chronic supplemental (100 ng/ml) level of folic acid to cells exposed to a normal (10 ng/ml) level of folic acid, in the presence of vitamin B12 and other micronutrients involved in the folate-methionine cycle. In addition to higher intracellular folate levels, HT29 cells at 100 ng folic acid/ml displayed faster growth and higher metabolic activity. cDNA microarray analysis indicated an effect on cell turnover and Fe metabolism. We fully confirmed these effects at the physiological level. At 100 ng/ml, cell assays showed higher proliferation and apoptosis, while gene expression analysis and a lower E-cadherin protein expression indicated decreased differentiation. These results are in agreement with the promoting effect of folic acid supplementation on established colorectal neoplasms. The lower expression of genes related to Fe metabolism at 100 ng folic acid/ml was confirmed by lower intracellular Fe levels in the cells exposed to folic acid at 100 ng/ml. This suggests an effect of folate on Fe metabolism.

Differential gene expression in intestinal epithelial cells induced by single and mixtures of potato glycoalkaloids.

Alpha-chaconine and alpha-solanine are naturally occurring toxins. They account for 95% of the total glycoalkaloids in potatoes ( Solanum tuberosum L.). At high levels, these glycoalkaloids may be toxic to humans, mainly by disrupting cell membranes of the gastrointestinal tract. Gene-profiling experiments were performed, whereby Caco-2 cells were exposed to equivalent concentrations (10 microM) of pure alpha-chaconine or alpha-solanine or glycoalkaloid mixtures of varying alpha-chaconine/alpha-solanine ratios for 6 h. In addition, lactate dehydrogenase, cell cycle, and apoptosis analyses experiments were also conducted to further elucidate the effects of glycoalkaloids. The main aims of the study were to determine the transcriptional effects of these glycoalkaloid treatments on Caco-2 cells and to investigate DNA microarray utility in conjunction with conventional toxicology in screening for potential toxicities and their severity. Gene expression and pathway analyses identified changes related to cholesterol biosynthesis, growth signaling, lipid and amino acid metabolism, mitogen-activated protein kinase (MAPK) and NF-kappaB cascades, cell cycle, and cell death/apoptosis. To varying extents, DNA microarrays discriminated the severity of the effect among the different glycoalkaloid treatments.

Induction of the cholesterol biosynthesis pathway in differentiated Caco-2 cells by the potato glycoalkaloid alpha-chaconine.

Glycoalkaloids are naturally occurring toxins in potatoes, which at high levels may induce toxic effects in humans, mainly on the gastrointestinal tract by cell membrane disruption. In order to better understand the molecular mechanisms underlying glycoalkaloid toxicity, we examined the effects of alpha-chaconine on gene expression in the Caco-2 intestinal epithelial cell line using DNA microarrays. Caco-2 cells were exposed for 6h to 10 microM alpha-chaconine in three independent experiments (randomized block design). The most prominent finding from our gene expression and pathway analyses was the upregulation of expression of several genes involved in cholesterol biosynthesis. This to some extent is in line with the literature-described mechanism of cell membrane disruption by glycoalkaloids. In addition, various growth factor signaling pathways were found to be significantly upregulated. This study is useful in understanding the mechanism(s) of alpha-chaconine toxicity, which may be extended to other potato glycoalkaloids more generally.

DNA microarray technology reveals similar gene expression patterns in rats with vitamin a deficiency and chemically induced colitis.

Previous studies suggest that vitamin A deficiency may induce or intensify inflammatory changes in the rat gastrointestinal system. The present study was designed to compare the expression profiles of rat models of vitamin A deficiency and induced colitis. cDNA-microarray technology was used to determine the genes involved in the inflammatory processes in the two models. mRNA was extracted from colons of rats that were vitamin A deficient, vitamin A supplemented (control), or had 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis, reverse-transcribed into fluorescence-labeled cDNA and hybridized onto microarrays containing a duplicate set of 1152 cDNAs, derived mainly from the colon carcinoma cell line Caco-2. Differential gene expression was detected in vitamin A deficiency and in TNBS-induced colitis vs. control. beta-Actin, translation initiation factor A4 and translation elongation factor 1, ornithine decarboxylase (ODC) and keratin 19 were markedly down-regulated, whereas spermidine/spermine N1-acetyltransferase (SSAT) and polyubiquitin (UbC) were up-regulated in both vitamin A-deficient rats and those with TNBS-induced colitis. The strong association between the differential gene expression in the two animal models, compared with the control, suggests that deficiency of vitamin A causes inflammatory changes in the rat colon that are similar to processes occurring in colitis. Further investigation is required to elucidate the importance of each of the regulated genes to the pathology of colitis and vitamin A inadequacy.