Luteolin protects against adipogenic and lipogenic potency induced by human relevant mixtures of persistent organic pollutants (POPs) in the 3T3-L1 model.

Human exposure to persistent organic pollutants (POPs) may contribute to obesogenic effects. We have previously shown that POP mixtures modelled on blood levels relevant to the Scandinavian population induces adipogenic effects in the mouse 3T3-L1 cell line. Luteolin is a flavone that has shown anti-lipogenic and anti-adipogenic effects on adipogenesis in in vitro models. In this study, luteolin has been applied to inhibit adipocyte formation and intracellular lipid content increase induced by a human relevant mixture of POPs. 3T3-L1 cells were exposed to a POP mixture consisting of 29 chemicals, including amongst others polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs), perfluoroalkylated acids (PFAAs), and polybrominated diphenyl ethers (PBDEs). Rosiglitazone was applied as a positive lipogenic control. Luteolin was tested between 0.5 and 10 µM. High content analysis was used to assess changes in adipocyte formation and intracellular lipid content in the 3T3-L1 cell line. Luteolin significantly reduced POP-induced adipocyte formation at 2, 5 and 10 µM, and lipid accumulation at 10 µM. Interestingly, luteolin did not affect rosiglitazone induced adipo- and lipogenic effects, suggesting differences in mechanisms of action. In conclusion, this in vitro study shows that dietary polyphenols such as luteolin may protect against POP induced adipo- and lipogenic effects.

A mixture of persistent organic pollutants relevant for human exposure inhibits the transactivation activity of the aryl hydrocarbon receptor in vitro.

While humans are exposed to mixtures of persistent organic pollutants (POPs), their risk assessment is usually based on a chemical-by-chemical approach. To assess the health effects associated with mixed exposures, knowledge on mixture toxicity is required. Several POPs are potential ligands of the Aryl hydrocarbon receptor (AhR), which involves in xenobiotic metabolism and controls many biological pathways. This study assesses AhR agonistic and antagonistic activities of 29 POPs individually and in mixtures by using Chemical-Activated LUciferase gene eXpression bioassays with 3 transgenic cell lines (rat hepatoma DR-H4IIE, human hepatoma DR-Hep G2 and human mammary gland carcinoma DR-T47-D). Among the 29 POPs, which were selected based on their abundance in Scandinavian human blood, only 4 exerted AhR agonistic activities, while 16 were AhR antagonists in DR-H4IIE, 5 in DR-Hep G2 and 7 in DR-T47-D when tested individually. The total POP mixture revealed to be AhR antagonistic. It antagonized EC50 TCDD inducing AhR transactivation at a concentration of 125 and 250 and 500 fold blood levels in DR-H4IIE, DR-T47-D and DR-Hep G2, respectively, although each compound was present at these concentrations lower than their LOEC values. Such values could occur in real-life in food contamination incidents or in exposed populations. In DR-H4IIE, the antagonism of the total POP mixture was due to chlorinated compounds and, in particular, to PCB-118 and PCB-138 which caused 90% of the antagonistic activity in the POP mixture. The 16 active AhR antagonists acted additively. Their mixed effect was predicted successfully by concentration addition or generalized concentration addition models, rather than independent action, with only two-fold IC50 underestimation. We also attained good predictions for the full dose-response curve of the antagonistic activity of the total POP mixture.

Human blood-based exposure levels of persistent organic pollutant (POP) mixtures antagonise androgen receptor transactivation and translocation.

INTRODUCTION: Human exposure to persistent organic pollutants (POPs) has been linked to genitourinary health-related conditions such as decreased sperm quality, hypospadias, and prostate cancer (PCa). Conventional risk assessment of POPs focuses on individual compounds. However, in real life, individuals are exposed to many compounds simultaneously. This might lead to combinatorial effects whereby the global effect of the mixture is different from the effect of the single elements or subgroups. POP mixtures may act as endocrine disruptors via the androgen receptor (AR) and potentially contribute to PCa development. AIM: To determine the endocrine disrupting activity of a POP mixture and sub-mixtures based upon exposure levels detected in a human Scandinavian population, on AR transactivation and translocation in vitro. MATERIALS AND METHODS: The Total POP mixture combined 29 chemicals modelled on the exposure profile of a Scandinavian population and 6 sub-mixtures: brominated (Br), chlorinated (Cl), Cl + Br, perfluorinated (PFAA), PFAA + Br, PFAA + Cl, ranging from 1/10× to 500× relative to what is found in human blood. Transactivation was measured by reporter gene assay (RGA) and translocation activity was measured by high content analysis (HCA), each using stably transfected AR model cell lines. RESULTS: No agonist activity in terms of transactivation and translocation was detected for any POP mixtures. In the presence of testosterone the Cl + Br mixture at 100× and 500× blood level antagonised AR transactivation, whereas the PFAA mixture at blood level increased AR transactivation (P < 0.05). In the presence of testosterone the Cl and PFAA + Br mixtures at 1/10×, 1×, and 50× blood level antagonised AR translocation (P < 0.05). CONCLUSION: Taken together, some combinations of POP mixtures can interfere with AR translocation. However, in the transactivation assay, these combinations did not affect gene transactivation. Other POP combinations were identified here as modulators of AR-induced gene transactivation without affecting AR translocation. Thus, to fully evaluate the effect of environmental toxins on AR signalling, both types of assays need to be applied.

Ultra-late recurrences of gastro-intestinal carcinoma after primary resection: the mechanism of dormancy.

Curative resection of limited gastro-intestinal carcinoma does not always mean curation with tumor-free long-term survival. We present two cases of ultra-late recurrence 14 years after initial treatment. In the first case a 50-year-old male underwent in 1997 a subtotal esophagectomy with tubulation of the stomach for a localized Barrett carcinoma. Postoperative staging showed a poorly differentiated adenocarcinoma, pT1N1 (stage IIB). In May 2011, 14 years after the initial resection, multiple bone metastases were diagnosed and a biopsy confirmed the poorly differentiated carcinoma with the same characteristics as the primary tumor. Investigations showed no evidence for a new primary tumor. The second case is a 52-year old man who underwent a low anterior resection for a small rectal cancer in 1997, histologically a well differentiated adenocarcinoma, stage IB (pT2NO). In December 2011 multiple metastases were diagnosed and a biopsy showed a metastasis from a mucinous carcinoma, suggestive for a colorectal carcinoma. There was also no evidence for a new primary tumor. Although the prognosis of limited esophageal and colorectal cancer is good, recurrence is always possible and an ultra-late recurrence may exceptionally occur. The mechanism of tumor dormancy is described.

Endocrine disrupting effects of ochratoxin A at the level of nuclear receptor activation and steroidogenesis.

Ochratoxin A (OTA) is a mycotoxin and extrolite of fungi which has been reported in a range of foods. This study uses mammalian reporter gene assays (RGAs) with natural steroid receptors and the H295R steroidogenesis assay to assess the endocrine disrupting activity of OTA. At the receptor level, OTA (within a concentration range of 0.25-2500 ng/ml) did not induce an agonistic response in an oestrogen, androgen, progestagen or glucocorticoid RGA. An antagonistic effect was observed in all of the RGAs at the highest concentration tested (2500 ng/ml). However, while there was no significant cytotoxic effect observed in the MTT (thiazolyl blue tetrazolium bromide) cell viability assay at this concentration, there was a corresponding change in cell morphology which may be related to the resulting antagonistic effect. At the hormone production level, H295R cells were used as a steroidogenesis model and exposed to OTA (within a concentration range of 0.1-1000 ng/ml). Treatment of the cells with 1000 ng/ml OTA increased the production of estradiol (117±14 ng/ml) over 3 times that of the solvent control (36±9 pg/ml). Western blotting confirmed an increase in aromatase protein. Overall the results indicate that OTA does not appear to interact with steroid receptors but has the potential to cause endocrine disruption by interfering with steroidogenesis. This is the first study identifying the effect OTA may have on production of the steroid hormone estradiol.

An in vitro investigation of endocrine disrupting effects of trichothecenes deoxynivalenol (DON), T-2 and HT-2 toxins.

Trichothecenes are a large family of chemically related mycotoxins. Deoxynivalenol (DON), T-2 and HT-2 toxins belong to this family and are produced by various species of Fusarium. The H295R steroidogenesis assay, regulation of steroidogenic gene expression and reporter gene assays (RGAs) for the detection of androgen, estrogen, progestagen and glucocorticoid (ant)agonist responses, have been used to assess the endocrine disrupting activity of DON, T-2 and HT-2 toxins. H295R cells were used as a model for steroidogenesis and gene expression studies and exposed with either DON (0.1-1000ng/ml), T-2 toxin (0.0005-5ng/ml) or HT-2 toxin (0.005-50ng/ml) for 48h. We observed a reduction in hormone levels in media of exposed cells following radioimmunoassay. Cell viability was determined by four colorimetric assays and we observed reduced cell viability with increasing toxin concentrations partly explaining the significant reduction in hormone levels at the highest toxin concentration of all three trichothecenes. Thirteen of the 16 steroidogenic genes analyzed by quantitative real time PCR (RT-qPCR) were significantly regulated (P<0.05) by DON (100ng/ml), T-2 toxin (0.5ng/ml) and HT-2 toxin (5ng/ml) compared to the control, with reference genes (B2M, ATP5B and ACTB). Whereas HMGR and CYP19 were down-regulated, CYP1A1 and CYP21 were up-regulated by all three trichothecenes. DON further up-regulated CYP17, HSD3B2, CYP11B2 and CYP11B1 and down-regulated NR5A1. T-2 toxin caused down-regulation of NR0B1 and NR5A1 whereas HT-2 toxin induced up-regulation of EPHX and HSD17B1 and down-regulation of CYP11A and CYP17. The expressions of MC2R, StAR and HSD17B4 genes were not significantly affected by any of the trichothecenes in the present study. Although the results indicate that there is no evidence to suggest that DON, T-2 and HT-2 toxins directly interact with the steroid hormone receptors to cause endocrine disruption, the present findings indicate that exposure to DON, T-2 toxin and HT-2 toxin have effects on cell viability, steroidogenesis and alteration in gene expression indicating their potential as endocrine disruptors.

Dysphagia lusoria caused by a right-sided aorta.

We report a case of an 80-year-old female with dysphagia lusoria caused by oesophageal compression by a right-sided atheromatous aorta. The relationship between aortic root vascular anomalies and dysphagia has been clearly established in literature and can be diagnosed by a barium swallowing study, followed by CT or MRI. Aortic anomalies and variations in aortic branches are caused by embryonic malformations and are mostly described in association with congenital heart lesions. In this pauci-symptomatic patient, the preferred treatment is a conservative management.

Endocrine disrupting effects of zearalenone, alpha- and beta-zearalenol at the level of nuclear receptor binding and steroidogenesis.

The mycotoxin zearalenone (ZEN) is a secondary metabolite of fungi which is produced by certain species of the genus Fusarium and can occur in cereals and other plant products. Reporter gene assays incorporating natural steroid receptors and the H295R steroidogenesis assay have been implemented to assess the endocrine disrupting activity of ZEN and its metabolites α-zearalenol (α-ZOL) and ß-zearalenol (ß-ZOL). α-ZOL exhibited the strongest estrogenic potency (EC(50) 0.022±0.001 nM), slightly less potent than 17-ß estradiol (EC(50) 0.015±0.002 nM). ZEN was ~70 times less potent than α-ZOL and twice as potent as ß-ZOL. Binding of progesterone to the progestagen receptor was shown to be synergistically increased in the presence of ZEN, α-ZOL or ß-ZOL. ZEN, α-ZOL or ß-ZOL increased production of progesterone, estradiol, testosterone and cortisol hormones in the H295R steroidogenesis assay, with peak productions at 10 µM. At 100 µM, cell viability decreased and levels of hormones were significantly reduced except for progesterone. ß-ZOL increased estradiol concentrations more than α-ZOL or ZEN, with a maximum effect at 10 µM, with ß-ZOL (562±59 pg/ml)>α-ZOL (494±60 pg/ml)>ZEN (375±43 pg/ml). The results indicate that ZEN and its metabolites can act as potential endocrine disruptors at the level of nuclear receptor signalling and by altering hormone production.

Steroidogenesis in primary cultures of neonatal porcine Leydig cells from Duroc and Norwegian Landrace breeds.

Breed differences in steroidogenic activity between primary Leydig cells derived from neonatal purebred Duroc and Norwegian Landrace boars were investigated in vitro. Concentrations of testosterone, estradiol, androstenone, cortisol and progesterone produced into the medium were determined. To explore underlying mechanisms the cellular expression of a suite of genes relevant in steroidogenesis was measured using reverse transcription and quantitative PCR (RT-qPCR). Basal steroid concentrations indicated a larger production capacity for steroids in unstimulated Duroc cells. Stimulation of the cells with LH increased steroid hormone secretion significantly in both breeds in a dose dependent manner. Testosterone and androstenone concentrations increased approximately 50- and 15-fold, respectively, whereas concentrations of estradiol, cortisol and progesterone increased to a lesser extent. At levels of maximal LH stimulation, absolute steroid concentrations were higher in Duroc. However, the relative increase in hormone concentrations was significantly lower in Duroc cells for estradiol, progesterone and cortisol when compared to basal levels. LH exposure was associated with a general up-regulation of mRNA levels for steroidogenic genes, stronger in Duroc than in Norwegian Landrace. This was in agreement with the higher absolute concentrations of steroid hormones measured in culture medium from the LH-stimulated Duroc Leydig cells, but did not concur with the fact that the relative increase in hormone production was lower in Duroc than in Norwegian Landrace Leydig cells for some hormones. It was concluded that breed differences in steroid hormone concentrations and gene expression between Norwegian Landrace and Duroc are complex and cannot be explained by a simple mechanism of action.

Effects of mixtures of persistent organic pollutants (POPs) derived from cod liver oil on H295R steroidogenesis.

Crude cod liver oil and liver oil supplements are consumed as a source of vitamin A, D and polyunsaturated fatty acids; during winter and early pregnancy. Crude cod liver oil however constitutes a considerable source of persistent organic pollutants (POPs). This paper aimed at characterizing and quantifying the influence of POP mixtures extracted from three different steps in the cod liver oil industrial process on hormone production and the expression of steroidogenesis-related genes in H295R cells. Exposure to extracts from crude cod liver oil and from its industrial waste increased progesterone (P4), cortisol (Cort), testosterone (T) and estradiol (E2) production; and among others, the expression of MC2R, CYP11B1 and HSD3B2 genes. Observed effects after exposure to pharmaceutical cod liver oil extract were considerably lower. The type of effects on gene expression and hormone production were similar to those induced by forskolin and PCBs, the latter being the major contaminants within the extracts. Additional research is required to further unveil the mechanisms behind the observed steroidogenic effects and to assess whether the potential risk might outweigh the potential benefits of crude and processed cod liver oil consumption.

Differential effects of antiepileptic drugs on steroidogenesis in a human in vitro cell model.

OBJECTIVES: To better understand the interaction of antiepileptic drugs and production of sex hormones, possible effects of valproate (VPA), levetiracetam (LEV) and carbamazepine (CBZ) on steroidogenesis were investigated in the human adrenal carcinoma cell line H295R. MATERIALS AND METHODS: H295R cells were exposed to different concentrations of VPA, LEV or CBZ for 48 h. Sex hormone concentrations and mRNA expression levels were analyzed via radioimmunoassay and quantitative real time (RT)-PCR, respectively. RESULTS: In VPA-exposed cells estradiol levels decreased in a dose-dependent manner, while testosterone and progesterone levels were unaffected. Expression of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR), steroidogenic acute regulatory protein (StAR), CYP11a, CYP17, CYP21, 3betaHSD2, 17betaHSD1 was downregulated and expression of CYP11beta2 was upregulated. No effect on sex hormone production was observed under influence of LEV or CBZ. Expression of StAR, CYP17, CYP19 and 3betaHSD2 was downregulated in LEV-exposed cells, and expression of HMGR, CYP11beta2 and CYP17 was downregulated in CBZ-exposed cells. CONCLUSIONS: VPA exposure resulted in a decrease in estradiol levels and a general downregulation of expression of genes encoding for enzymes early in steroidogenesis. No consistent changes were seen with LEV or CBZ exposure.

Esophageal lipomatosis: another consequence of the use of steroids.

After we incidentally found on CT extensive esophageal fat accumulations in a patient with long-term use of steroids, we prospectively evaluated during a 6-month period all CT studies of the chest for esophageal lipomatosis and related the findings to the possible use of steroids. The diagnosis of esophageal fat on CT was made by density measurements or if too small for reliable density measurements by comparison with mediastinal fat. In 21 of 1,320 exclusively older male patients the diagnosis of esophageal lipomatosis was definite in 7 and likely in 14 patients. All fat accumulations were located in the upper third of the esophagus (mean length 22 +/- 6 mm) and presented ring-like (n = 10), irregular (n = 3), or as a horseshoe sparing the posterior border (n = 8). In 20 patients there was an unequivocal history of steroid treatment. Associated centripetal fat infiltration was found in 11 patients. None of the patients had swallowing problems. Prolonged use of steroids, either orally or inhalationally administered, is associated with esophageal lipomatosis. The predisposition for the upper esophagus might be related to the presence of striated muscle cells in this part of the esophagus; moreover, inhalational steroid therapy may adversely affect the upper esophagus.

Culture filtrates of Aspergillus fumigatus induce different modes of cell death in human cancer cell lines.

Aspergillus fumigatus culture filtrate (CF) has a potent cytotoxic effect on three human cancer cell lines (DLKP, A549 and HEp-2) and initiates cell death by apoptosis but the execution of the apoptotic process is incomplete. DLKP cells treated with A. fumigatus CF demonstrate features associated with apoptosis but cytoplasmic and nuclear fragmentation were not observed and cells ultimately underwent necrosis. The apoptotic process commenced in A549 and HEp-2 cells upon exposure to CF, cell shrinkage was observed but membrane blebbing and apoptotic body formation were not detected and detached cells died by necrosis. In contrast, extensive nuclear fragmentation and apoptotic body formation were evident in DLKP and A549 cells treated with anti-neoplastic agents. This work indicates that A. fumigatus CF is cytotoxic to cancer cells and can initiate apoptosis but that the complete apoptotic pathway is not followed.

Enhancement of chemotherapeutic drug toxicity to human tumour cells in vitro by a subset of non-steroidal anti-inflammatory drugs (NSAIDs).

The effect on cytotoxicity of combining a range of clinically important non-steroidal anti-inflammatory drugs (NSAIDs) with a variety of chemotherapeutic drugs was examined in the human lung cancer cell lines DLKP, A549, COR L23P and COR L23R and in a human leukaemia line HL60/ADR. A specific group of NSAIDs (indomethacin, sulindac, tolmetin, acemetacin, zomepirac and mefenamic acid) all at non-toxic levels, significantly increased the cytotoxicity of the anthracyclines (doxorubicin, daunorubicin and epirubicin), as well as teniposide, VP-16 and vincristine, but not the other vinca alkaloids vinblastine and vinorelbine. A substantial number of other anticancer drugs, including methotrexate, 5-fluorouracil, cytarabine, hydroxyurea, chlorambucil, cyclophosphamide, cisplatin, carboplatin, mitoxantrone, actinomycin D, bleomycin, paclitaxel and camptothecin, were also tested, but displayed no synergy in combination with the NSAIDs. The synergistic effect was concentration dependent. The effect appears to be independent of the cyclo-oxygenase inhibitory ability of the NSAIDs, as (i) the synergistic combination could not be reversed by the addition of prostaglandins D2 or E2; (ii) sulindac sulphone, a metabolite of sulindac that does not inhibit the cyclooxygenase enzyme, was positive in the combination assay: and (iii) many NSAIDs known to be cyclo-oxygenase inhibitors, e.g. meclofenamic acid, diclofenac, naproxen, fenoprofen, phenylbutazone, flufenamic acid, flurbiprofen, ibuprofen and ketoprofen, were inactive in the combination assay. The enhancement of cytotoxicity was observed in a range of drug sensitive tumour cell lines, but did not occur in P-170-overexpressing multidrug resistant cell lines. However, in the HL60/ADR and COR L23R cell lines, in which multidrug resistance is due to overexpression of the multidrug resistance-associated protein MRP, a significant increase in cytotoxicity was observed in the presence of the active NSAIDs. Subsequent Western blot analysis of the drug sensitive parental cell lines, DLKP and A549, revealed that they also expressed MRP and reverse-transcription-polymerase chain reaction studies demonstrated that mRNA for MRP was present in both cell lines. It was found that the positive NSAIDs were among the more potent inhibitors of [3H]-LTC4 transport into inside-out plasma membrane vesicles prepared from MRP-expressing cells, of doxorubicin efflux from preloaded cells and of glutathione-S-transferase activity. The NSAIDs did not enhance cellular sensitivity to radiation. The combination of specific NSAIDs with anticancer drugs reported here may have potential clinical applications, especially in the circumvention of MRP-mediated multidrug resistance.

Membrane changes associated with the early stages of apoptosis in HEp-2 cells decrease susceptibility to adherence by Candida albicans.

The aim of the present work was to establish whether apoptotic HEp-2 cells demonstrated an altered susceptibility to adherence by the pathogenic yeast Candida albicans. Cultures of HEp-2 cells were treated with concentrations of three chemotherapeutic agents sufficient to induce cell death by apoptosis and this was confirmed by microscopic examination and by the loss of membrane asymmetry (as indicated by phosphatidylserine externalization) and the fragmentation of nuclear DNA into distinct subunits. Cells in the early stages of apoptosis demonstrated a reduced susceptibility to adherence by a number of strains of C. albicans. A correlation between the decrease in yeast adherence and the loss of membrane asymmetry, associated with the early stages of apoptosis, was established.

Inhibition of apoptosis by antioxidants in the human HL-60 leukemia cell line.

Cell death via apoptosis is an important event involved in a number of immunological processes. Recently, apoptosis has been associated with oxidative stress in a number of cell systems. Here we assessed the inhibitory capacity of different antioxidants on UV- and drug-induced apoptosis in the human leukemic cell line, HL-60. We found that the oxygen radical scavenger, BHA, the radioprotector cysteamine and the metal chelators, pyrrolidinedithiocarbamate (PDTC), diethyldithiocarbamate (DEDTC), and dimethyldithiocarbamate (DMDTC), were able to significantly inhibit nuclear fragmentation and reduce the formation of apoptotic bodies in UV-irradiated human leukemic cells. Both BHA and PDTC were found to reduce DNA fragmentation as assessed by in situ DNA nick-end labelling and quantification thereof using fluorescence flow cytometry. In addition to inhibiting UV-induced apoptosis, PDTC was also capable of reducing the amount of apoptosis induced by a range of cytotoxic drugs, such as actinomycin-D, camptothecin, etoposide, and melphalan, whereas BHA and cysteamine were not as effective in these cases after more than four hours in culture when compared to PDTC. To further elucidate the working mechanism of PDTC, we have looked at the effect of PDTC on DNA fragmentation in isolated nuclei, under conditions that promote activation of endogenous endonuclease involved in apoptosis. In contrast to ZnCl2, a potent inhibitor of endonuclease activity, PDTC was unable to inhibit DNA-ladder formation in this assay. Taken together, these results indicate that oxygen radicals may have a central role to play in the induction of apoptosis and that dithiocarbamates can serve as potent inhibitors of apoptosis induced by a wide variety of stimuli.

Zinc inhibits UV radiation-induced apoptosis but fails to prevent subsequent cell death.

Zinc ions inhibit the morphological and DNA fragmentation features of apoptosis in a number of systems. HL-60 cells pretreated with zinc and exposed to UV irradiation maintained their normal morphology for up to 8 h, whereas non-zinc-treated cells underwent extensive apoptosis. Zinc pretreatment also inhibited both single and double-stranded DNA fragmentation, which is characteristic of apoptosis. The most effective zinc concentration that blocked apoptosis over short incubation periods (up to 8 h) was also the most toxic over extended time periods (> or = 12 h). The mechanism of cell death at these longer time periods was akin to necrosis, but occurred in the absence of any DNA fragmentation. The effects of the nuclease inhibitor aurintricarboxylic acid (ATA) was also examined on UV-induced apoptosis in HL-60 cells. ATA had no toxic effects over the concentration range tested, but also failed to prevent DNA fragmentation in whole cells. Further analysis showed that it effectively inhibited DNA fragmentation in isolated nuclei.