In vitro differential responses of rat and human aryl hydrocarbon receptor to two distinct ligands and to different polyphenols.

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) and several other environment/food-borne toxic compounds induce their toxicity via the aryl hydrocarbon receptor (AhR). AhR is also modulated by various endogenous ligands e.g. highly potent tryptophan (Trp)-derivative FICZ (6-formylindolo[3,2-b]carbazole) and natural ligands abundant in the human diet e.g. polyphenols. Therefore, evaluating AhR species-specific responses is crucial for understanding AhR physiological functions, establishing risk assessments, and exploring the applicability of AhR mediators in drug and food industry towards human-based usages. We studied AhR transactivation of FICZ/TCDD in vitro in a time-dependent and species-specific manner using dioxin responsive luciferase reporter gene assays derived from rat (DR-H4IIE) and human (DR-HepG2) hepatoma cells. We observed for the first time that FICZ potency was similar in both cell lines and was 40 times higher than TCDD in DR-HepG2 cells. Depleting Trp-derivative endogenously produced ligands by using culture medium without Trp, resulted in 3-fold higher AhR activation upon adding FICZ in DR-H4IIE cells, in contrast to DR-HepG2 cells which revealed a fast degradation of FICZ induction from 10 h post-exposure to complete disappearance after 24 h. Seven polyphenols and a mixture thereof, chosen based on commercially recommended doses and adjusted to human realistic exposure, caused rat and human species-specific AhR responses. Two isoflavones (daidzein and genistein) induced rat AhR synergistic effects with FICZ and/or TCDD, while quercetin, chrysin, curcumin, resveratrol, and the mixture exerted a strong inhibitory effect on the human AhR. Strikingly, resveratrol and quercetin at their realistic nanomolar concentrations acted additively in the mixture to abolish human AhR activation induced by various TCDD concentrations. Taken together, these results illustrate the species-specific complexity of AhR transcriptional activities modulated by various ligands and highlight the need for studies of human-based approaches.

Elucidating the aryl hydrocarbon receptor antagonism from a chemical-structural perspective.

The aryl hydrocarbon receptor (AhR) plays an important role in several biological processes such as reproduction, immunity and homoeostasis. However, little is known on the chemical-structural and physicochemical features that influence the activity of AhR antagonistic modulators. In the present report, in vitro AhR antagonistic activity evaluations, based on a chemical-activated luciferase gene expression (AhR-CALUX) bioassay, and an extensive literature review were performed with the aim of constructing a structurally diverse database of contaminants and potentially toxic chemicals. Subsequently, QSAR models based on Linear Discriminant Analysis and Logistic Regression, as well as two toxicophoric hypotheses were proposed to model the AhR antagonistic activity of the built dataset. The QSAR models were rigorously validated yielding satisfactory performance for all classification parameters. Likewise, the toxicophoric hypotheses were validated using a diverse set of 350 decoys, demonstrating adequate robustness and predictive power. Chemical interpretations of both the QSAR and toxicophoric models suggested that hydrophobic constraints, the presence of aromatic rings and electron-acceptor moieties are critical for the AhR antagonism. Therefore, it is hoped that the deductions obtained in the present study will contribute to elucidate further on the structural and physicochemical factors influencing the AhR antagonistic activity of chemical compounds.

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.

Assessment of methods of detection of water estrogenicity for their use as monitoring tools in a process of estrogenicity removal.

Methods of monitoring of estrogenicity in water were gathered, compared, and tested within the context of their practical use as measurement and design tools, in the development of a process of degradation of estrogenic endocrine disruptors. In this work, the focus was put on in vitro assays, with the use of analytical techniques as additional analysis when possible. Practically, from a literature review, four methods that seemed most suitable to practical use required in a process development were tested: the Yeast Estrogen Screen assay, the Lyticase-assisted Yeast Estrogen Screen assay (LYES), the MMV-LUC assay and the HPLC-UV analytical method. Dose-response curves in response to estrogenic standard 17ß-estradiol were compared. Bisphenol A estrogenicity was measured by the methods as well. The model for the calculation of estradiol equivalents as measurements units was adapted. The methods were assessed in terms of ranges of detection, time of experiment, cost, ease of the experiment, reproducibility, etc. Based on that assessment, the LYES assay was selected and successfully applied to the monitoring of estrogenicity removal from 17ß-estradiol and bisphenol A. More precisely, the bioassay allowed the acquisition of kinetic curves for a laboratory-scaled process of estrogenicity removal by immobilized enzymes in a continuous packed-bed reactor. The LYES assay was found to have a real methodological potential for scale-up and design of a treatment process. The HPLC-UV method showed good complementarity with the LYES assay for the monitoring of bisphenol A concentrations in parallel with estrogenicity, reporting no significant estrogenicity from degradation byproducts, among others.

Estimation of furan contamination across the Belgian food chain.

This paper provides an estimate of the furan content of Belgian foods. The objective of the study was to achieve the best food chain coverage with a restricted number of samples (n = 496). The geographic distribution, different market chains and labels, and consumption frequencies were taken into account in the construction of the sampling plan. Weighting factors such as contamination levels, consumption frequency and the diversity of food items were applied to set up the model. The very low detection capabilities (CC(ß)) of the analytical methods used (sub-ppb) allowed reporting of 78.2% of the overall dataset above CC(ß) and, in particular, 96.7% for the baby food category. The highest furan levels were found in powdered roasted bean coffee (1912 µg kg(-1)) with a mean of 756 µg kg(-1) for this category. Prepared meat, pasta and rice, breakfast cereals, soups, and baby food also showed high mean furan contents ranging from 16 to 43 µg kg(-1). Comparisons with contamination surveys carried out in other countries pointed out differences for the same food group and therefore contamination levels are related to the geographical origin of food items.

Potential of an in vitro toolbox combined with exposure data as a first step for the risk assessment of dietary chemical contaminants.

In vitro risk assessment of dietary contaminants has become a priority in human food safety. This paper proposes an in vitro approach associating different complementary tools in an original toolbox and aims to improve the assessment of the toxicological impact of dietary contaminants at realistic human exposure levels, with a special focus on the intestinal compartment. The system is based on the use of four complementary cellular tools, namely stress gene induction in transgenic strains of Escherichia coli, modulation of the activity of key biotransformation enzymes (cytochrome P-450 (CYP) 1A1 and 3A4) in a human intestinal cell line, and activation of aryl hydrocarbon receptor (AhR) and oestrogenic receptor (ER)-dependent genes in agonistic and antagonistic assays with luciferase reporter cells. It was applied to four chosen model molecules: ochratoxin A (OTA) and deoxynivalenol (DON), two common food-borne mycotoxins, and imazalil (IMA) and benomyl (BEN), two fungicides widely occurring in foodstuffs. All these assays were performed at or around a realistic intestinal concentration, determined through a deterministic approach based on the calculation of a theoretical maximum daily intake (TMDI). Using the four model molecules, it is clearly highlighted that induction of CYP1A1 activity and inhibition of CYP3A4 activity occurred in Caco-2 cells at a realistic intestinal concentration of IMA. Furthermore, some bacterial stress genes were induced in a range of realistic concentrations, following exposure to DON and IMA. In addition, BEN clearly provoked an ER agonistic activity in a human oestrogen sensitive reporter cell line. All these results are in accordance with the literature, suggesting that the in vitro toolbox constitutes an interesting approach in order to obtain a first 'fingerprint' of dietary contaminants at realistic human exposure for further risk assessment.

Modulation of CYP1A1 activity by a Ginkgo biloba extract in the human intestinal Caco-2 cells.

Ginkgo biloba is a widely consumed dietary supplement. Some dietary active compounds modulate the activity of biotransformation enzymes inside the enterocytes and more interestingly of cytochrome P-450 1A1 (CYP1A1). This enzyme is of a particular interest because of its implication in the metabolism of some exogenous pro-carcinogens or endogenous molecules. In the present work, we have used Caco-2 cells to study the effect of a standard reference material of a Ginkgo biloba extract (GBE) (10-400 µg/ml), as well as of its major individual active compounds (kaempferol, quercetin, isorhamnetin, ginkgolides and bilobalide), alone or in mixtures, at realistic intestinal concentrations, on the induction of CYP1A1 activity, in the presence or absence of benzo[a]pyrene (B[a]P) (0.1 µg/ml), a well-known CYP1A1 inducer. 3-O-rutinosides of kaempferol, quercetin and isorhamnetin were also tested. We have demonstrated a strong induction (p < 0.005) of CYP1A1 activity and a slight, but significant (p < 0.005), decrease of this activity in the presence of B[a]P by the GBE at the realistic exposure level of 100 µg/ml. The inductive effect was explained, in part, by quercetin and kaempferol after 24h exposure while unknown compounds seem to be responsible for the strong CYP1A1 induction observed after 6h exposure. The inhibitory potency of flavonols on CYP1A1 activity in presence of B[a]P was much stronger for the aglycones than for the 3-O-rutinosides, explaining the slight effect observed with the GBE, mainly composed of glycosylated flavonoids. These results indicate that GBEs may disturb intestinal CYP1A1 activity and, in turn, affect the metabolism of other compounds. The present paper thus highlights the necessity to take these side effects into account when administrating Ginkgo biloba herbal supplements.

Control of the illegal administration of natural steroid hormones in the plasma of bulls and heifers.

In the context of the control of the illegal administration of natural steroid hormones in cattle husbandry, an attempt was made to establish the decision levels for sex steroid hormones in the plasma of adult cattle, taking into account the effect of the treatment. Bulls and heifers were treated with two injections, at a two week interval, of an estradiol-testosterone cocktail. Steroid hormone and biochemical precursor concentrations were measured in plasma samples by using specific radioimmunoassays, before and after the treatment. When the treatment significantly (p < 0.05) modified a hormone concentration, a decision level was established for that hormone concentration. At each decision level, a score was assigned that represented the percentage of treated animals detected when the decision limit was applied. For heifers, 17 beta-estradiol and testosterone concentrations in plasma, which increased after the treatment, are the best criteria to use to detect treated animals, with decision limits of 20 pg ml-1 and 125 pg ml-1, respectively. In the instance of bulls, both testosterone and steroid biochemical precursor concentrations decreased in the plasma after the treatment. We proposed decision limits of 1500 pg ml-1 and 28 pg ml-1 for testosterone and androstenedione concentrations, respectively, the bulls displaying concentrations below these limits being positive. We observed that the repetition of the injection increased the score of the decision limit. The scores for testosterone are 70%, 14d after the first injection and 100% 14 d after the second injection, and for androstenedione, these scores are 60 and 100%, respectively.

Increase in epidermal growth factor receptor and its mRNA levels by parathyroid hormone (1-34) and parathyroid hormone-related protein (1-34) during differentiation of human trophoblast cells in culture.

Human cytotrophoblasts in culture aggregate and fuse to form syncytiotrophoblasts. This process is associated with an increase in epidermal growth factor receptor (EGFR) expression [Alsat et al.: J Cell Physiol 154:122-128, 1993]. Recent studies have demonstrated the presence of parathyroid hormone-related protein (PTHrP) in the human uterus and placenta. This led us to study the effect of PTH (1-34) and PTHrP (1-34) on the expression of EGFR during this differentiation process. Both peptides induced a concentration-dependent increase in EGF binding, with a maximal effect at the physiological concentration of 1 nM. EGFR protein level assessed by cross-linking and immunoblotting and EGFR biological activity assessed by measuring its EGF-induced autophosphorylation were increased 2- and 2.5-fold, respectively, when cells were treated for 24 h with 0.1 microM PTHrP or PTH compared to control cells. This effect was time-dependent with a maximum at 3 h of treatment. This treatment also increased trophoblast cell EGFR mRNA levels, suggesting transcriptional regulation of the EGFR. To ascertain whether activation of protein kinase C (PKC) or protein kinase A (PKA) is involved in this PTH effect, we determined EGFR protein level and EGFR autophosphorylation after exposure of cells to PKA inhibitor and PKC inhibitor, alone or together with the peptide. The presence of a PKC inhibitor blocked a further increase in EGFR number by PTH, while PKA inhibitor had no effect. These results show that PTH and PTHrP increase the synthesis of EGF receptors which are strongly expressed in syncytiotrophoblasts and suggested that these peptides might be involved in human placental development.

Adenohypophysis hormone gene products in 14 pituitary adenomas: analysis by immunohistochemistry and northern blotting.

We investigated 14 pituitary adenomas (10 silent adenomas; 3 prolactinomas and one GH-secreting tumor) for the presence of hormone gene transcripts (Northern blot) as well as for translation products (immunohistochemistry). The GH-secreting tumor was shown to express the genes coding for GH and PRL and to synthesize the corresponding hormones. In the cases of prolactinomas, immunohistochemical data demonstrated the synthesis of prolactin only. In addition to the PRL gene, Northern blot analysis revealed the transcription of the alpha-subunit gene in one case. Hormone genes were found to be expressed in 7 out of the 10 silent tumors, whereas no hormone synthesis was detected in any of these tissues. LH-beta mRNA was found in 3 cases, FSH-beta mRNA in 5 cases and alpha-subunit gene was shown to be expressed in one case. Surprisingly, the level of expression of the FSH-beta gene was higher than in normal tissue. This study confirms that some so called "silent" adenomas are expressing alpha- and/or beta-subunit glycoprotein hormone genes, even if no hormone is synthesized. The therapeutic action of bromocriptine described in some "silent" adenomas cases could be related to that hormone gene expression potentiality.

Regulation of growth hormone secretion in human trophoblastic cells in culture.

In this study, we have cultured in vitro purified trophoblastic cells from first-trimester and term human placenta. These cells were obtained by specific enzymatic digestion and centrifugation through a Percoll gradient. Using 2 specific monoclonal antibodies, the pituitary 22-kD growth hormone (GH) and the placental GH variant were assayed in the culture medium by radioimmunoassay. After 48 h of culture, only the placental GH variant was measured in the medium corresponding to first-trimester placenta (3.4 ng/24 h/10(5) cells). Surprisingly, an immunoactivity pattern of pituitary GH type was found in 3 out of 5 media conditioned with term placenta cells, while GH immunoactivity was very low, around the detection level, in the 2 others. These secretions are not modified with the time in culture and the state of differentiation of the cells from cytotrophoblast to syncytiotrophoblast. Neither in early nor in term placenta does the addition of GH-releasing factor (10(-6) M in the culture medium) stimulate the secretion of pituitary 22-kD GH or placental GH variant.

A non-radioactive method to detect RNA or DNA using an oligonucleotide probe with bromodeoxyuridine free ends, a monoclonal antibody against bromodeoxyuridine and immunogold silver staining.

A non radioactive method for probing RNA or DNA on dot and Northern blots using a synthetic oligonucleotide with bromodeoxyuridine free ends is described. The present experiment was carried out with human testis and placental RNA's. The probe was the 21 base long sequence coding for the amino acids 18 to 24 of the insulin-like growth factor I (IGF-I) with two bromodeoxyuridine dinucleotides added at the 5' and 3' ends. The probe was detected with a monoclonal antibody against bromodeoxyuridine and immunogold silver staining (IGSS). Our method was compared to the peroxydase (HRP) revelation of the same probe. The results obtained show a lower background with IGSS than with HRP revelation. A sensitivity similar to that of 32P labelling was found with the advantages of an increase in the rapidity of the procedure (24 hours instead of 9 days exposure) and the absence of handling radioactive substances. Moreover, as the monoclonal antibody against BrdU detects single stranded DNA only, the use of BrdU free ends-labelled oligonucleotide allows the development of the revelation procedure without any previous denaturation of the hybrid. This particular point is an indisputable advantage for detecting hybridization in situ.

Expression of the growth hormone variant gene in human placenta.

Besides the hGH-N gene, which codes for the pituitary 22 and 20K GH variants, the human genome contains a second GH gene, namely the GH-V, which has been thought to be silent. We recently discovered a placental variant of human growth hormone (hPGH), which appears in maternal serum at mid-pregnancy and which rises in concentration thereafter to term. As hPGH and GH-V proteins display very similar characteristics, including a high affinity for hepatic GH receptors, they could be identical. To verify this hypothesis, we sought hGH-V mRNA in placenta. Hybridization experiments were performed between dot-blotted mRNA originating either from placenta or from one pituitary hGH secreting adenoma and synthetic polynucleotide probes corresponding to specific portions of the hGH-V or hGH-N gene sequences. The results indicate that the V gene is indeed expressed in the placenta and, at a very low level, in the pituitary adenoma. Therefore hPGH is most likely the expression product of the hGH-V gene.

[Localization of transcription regulatory sequences. Application to the genes of the prolactin family]. / Localisation des séquences régulatrices de la transcription. Application aux gènes de la famille de la prolactine.

We are studying nucleotide sequences responsible for the regulation of eukaryotic gene expression. Our test system comprises the human genes coding for prolactin (hPRL), growth hormone (hGH-N) and placental lactogen (hCS-B). We have cloned these genes and are searching within their sequences for in vitro binding sites of the human glucocorticoid receptor on the hGH-N and hCS-B genes; the in vivo activity of such DNA sequences by assaying hybrid gene expression in transfected cells; in vivo "enhancer" activity of different hPRL gene fragments linked to a marker gene and transfected in cultured cells.