Anne Hillenweck

Characterization of Novel Ligands of ERα, Erβ, and PPARγ: The Case of Halogenated Bisphenol A and Their Conjugated Metabolites

The capability of the flame retardants tetrabromobisphenol A (TBBPA) and tetrachlorobisphenol A (TCBPA) to activate peroxysome proliferator-activated receptors (PPARs) α, β, and γ and estrogen receptors (ERs) α and β has been recently investigated, but the activity of their biotransformation products and of their lower molecular weight analogues formed in the environment remains unexplored. The aim of this study was to investigate the relationship between the degree of halogenation of BPA analogues and their affinity and activity towards human PPARγ and ERs and to characterize active metabolites of major marketed halogenated bisphenols. The biological activity of all compounds was studied using reporter cell lines expressing these nuclear receptors (NRs). We used NR-based affinity columns to rapidly evaluate the binding affinity of halogenated bisphenols for PPARγ and ERs and to trap active metabolites of TBBPA and TCBPA formed in HepG2 cells. The agonistic potential of BPA analogs highly depends on their halogenation degree: the bulkier halogenated BPA analogs, the greater their capability to activate PPARγ. In addition, PPARγ-based affinity column, HGELN-PPARγ reporter cell line and crystallographic analysis clearly demonstrate that the sulfation pathway, usually considered as a detoxification process, leads for TBBPA and TCBPA, to the formation of sulfate conjugates which possess a residual PPARγ-binding activity. Our results highlight the effectiveness NR-based affinity columns to trap and characterize biologically active compounds from complex matrices. Polyhalogenated bisphenols, but also some of their metabolites, are potential disrupters of PPARγ activity.

Use of the γH2AX assay for assessing the genotoxicity of polycyclic aromatic hydrocarbons in human cell lines

The development of in vitro genotoxic assays as an alternative method to animal experimentation is of growing interest in the context of the implementation of new regulations on chemicals. However, extrapolation of toxicity data from in vitro systems to in vivo models is hampered by the fact that in vitro systems vary in their capability to metabolize chemicals, and that biotransformation can greatly influence the experimental results. Therefore, much attention has to be paid to the cellular models used and experimental conditions. Polycyclic aromatic hydrocarbons (PAHs) are carcinogenic ubiquitous pollutants. Human exposure to PAHs is mainly from food origin. In this study, a detailed analysis of the biotransformation capabilities of three human cell lines commonly used for in vitro testing (HepG2, ACHN and Caco-2) was undertaken using 3 model PAHs (benzo(a)pyrene [B(a)P], fluoranthene [FLA] and 3-methylcholanthrene [3-MC]). Concomitantly the genotoxicity of these PAHs was investigated in different cell lines, using a new genotoxic assay (H2AX) in 96-well plates. The metabolic rates of B(a)P, FLA and 3-MC were similar in HepG2 and Caco-2 cell lines, respectively, though with the production of different metabolites. The ACHN cell line was shown to express very limited metabolic capabilities. We demonstrated that the PAHs having a high metabolic rate (B(a)P and 3-MC) were genotoxic from 10(-7) molar in both HepG2 and Caco-2 cells. The present study shows that H2AX measurement in human cell lines competent for the metabolism, is an efficient and sensitive genotoxic assay requiring less cells and time than other currently available tests.

3-Methylcholanthrene Displays Dual Effects on Estrogen Receptor (ER) α and ERβ Signaling in a Cell-Type Specific Fashion

The biological effects of 17β-estradiol (E2) are mediated by the two estrogen receptor (ER) isoforms ERα and ERβ. These receptors are ligand-inducible transcription factors that belong to the nuclear receptor superfamily. These receptors are also targets for a broad range of natural and synthetic compounds that induce ER activity, including dietary compounds, pharmaceuticals, and various types of environmental pollutants such as bisphenols and polychlorinated hydroxy-biphenyls. Here, we study the effect of the combustion byproduct 3-methylcholanthrene (3-MC) on ERα and ERβ. 3-MC is a compound identified previously as an activator of the aryl hydrocarbon receptor (AhR). Activation of AhR is traditionally associated with an inhibition of the E2 signaling network. In this study, we demonstrate that 3-MC is a cell-specific activator or inhibitor of E2 signaling pathways. We show that 3-MC acts as a repressor in some cells, presumably via the AhR, whereas it is a potent activator of ER activity in other cells. It is interesting that we demonstrate that the estrogenic effects of 3-MC are dependent on the ability of cells to metabolize parental 3-MC to alternative compounds. In summary, our results suggest that exposure to AhR ligands like 3-MC can lead to either activation or repression of E2 signaling, depending on the cellular context.

Use of reconstituted metabolic networks to assist in metabolomic data visualization and mining.

Metabolomics experiments seldom achieve their aim of comprehensively covering the entire metabolome. However, important information can be gleaned even from sparse datasets, which can be facilitated by placing the results within the context of known metabolic networks. Here we present a method that allows the automatic assignment of identified metabolites to positions within known metabolic networks, and, furthermore, allows automated extraction of sub-networks of biological significance. This latter feature is possible by use of a gap-filling algorithm. The utility of the algorithm in reconstructing and mining of metabolomics data is shown on two independent datasets generated with LC–MS LTQ-Orbitrap mass spectrometry. Biologically relevant metabolic sub-networks were extracted from both datasets. Moreover, a number of metabolites, whose presence eluded automatic selection within mass spectra, could be identified retrospectively by virtue of their inferred presence through gap filling.

Biotransformation of pentachlorophenol, aniline and biphenyl in isolated rainbow trout (Oncorhynchus mykiss) hepatocytes: comparison with in vivo metabolism

1. The biotransformation of pentachlorophenol (PCP), aniline and biphenyl in rainbow trout (Oncorhynchus mykiss) isolated liver cells was investigated to examine if fish hepatocytes represent a suitable alternative to the in vivo approach for studying the biotransformation of chemicals. Each compound was incubated at two concentrations (10 and 60 microM) for 2 h. For comparison, the metabolic profile of these xenobiotics was also studied in urine and bile of trout orally exposed to 1.8-4.0 mg/kg wet wt of each compound. 2. In vitro as in vivo, PCP glucuronide and to a lesser extent PCP sulphate were the metabolites formed by trout from PCP. 3. Aniline was mainly metabolized to acetanilide and to a lesser extent to 2-aminophenol by isolated hepatocytes, but neither hydroxylated acetanilide nor conjugates were found in vitro whereas they were present in bile and urine of trout treated with this chemical. 4. Trout hepatocytes metabolized biphenyl to hydroxylated and dihydroxylated products and the corresponding glucuronides. These results correlated well with the metabolic profile obtained from the bile of trout exposed to this pesticide. 5. It is concluded that although hepatocytes are well suited for several types of biotransformation studies, the fact that this system may in some cases produce a different metabolic pattern than in vivo should be considered when attempting to extrapolate in vitro to in vivo data.

Effects of diquat and fomesafen applied alone and in combination with a nonylphenol polyethoxylate adjuvant on Lemna minor in aquatic indoor microcosms.

The influence of tank-mix adjuvants on pesticide toxicity remains largely unknown. Agral 90, a nonylphenol polyethoxylated tank-mix adjuvant, has been used with diquat (bipyridylium herbicide) and fomesafen (diphenyl-ether herbicide) in aquatic indoor microcosms in order to compare the toxicity of the single compounds and of binary herbicide-adjuvant mixtures to Lemna minor. Twenty-four microcosms were used and treatments were performed with substances alone or with herbicide-adjuvant binary mixtures, at two concentrations levels (44.4 and 222.2 microg/L for the herbicides, and 100 and 500 microg/L for Agral 90). Toxicity was assessed weekly for 1 month through growth measurements, as inferred from the relative frond number (RFN) and relative frond area (RFA). Concentrations of diquat and fomesafen in water and sediments were measured weekly. The herbicides showed very different behaviour in microcosms, with a rapid disappearance of diquat from the aqueous phase whereas fomesafen levels remained almost constant over time. Diquat strongly inhibited the growth of L. minor whereas fomesafen had no effect on plant growth. Presence of the adjuvant only slightly reduced the effect of the lowest concentration of diquat, probably as a result of dispersion of the herbicide at the water surface. It is concluded that tank-mix adjuvant designed to improve herbicide efficiency in the terrestrial environment did not have any effect on aquatic plants when applied to the aquatic environment.

Characterization of biliary metabolites of fluoranthene in the common sole (Solea solea)

Fluoranthene is one of the most abundant polycyclic aromatic hydrocarbon (PAH) pollutants in the environment. Studies of the metabolism of PAHs have highlighted the importance of the gallbladder in concentrating xenobiotics in fish before excretion in feces. Analysis of bile metabolites can be considered useful for monitoring and assessing the exposure of fish to PAHs. Although the fate of several PAHs in marine organisms has been widely investigated, information is lacking regarding the metabolism of fluoranthene in fish. Therefore, we investigated the metabolic pathways of [14C]fluoranthene in the common sole (Solea solea) by identifying bile metabolites using electrospray ionization/mass spectrometry (ESI/MS) and nuclear magnetic resonance (NMR) spectroscopy. [14C]Fluoranthene was administered by intraperitoneal injection to 20 common soles. Groups of animals (n = 5) were killed 1, 2, 3, and 4 d postdosing, and gallbladders were excised for radioactivity counting and bile analysis. Biliary metabolites were separated and quantified by radio-high-performance liquid chromatography, and structure identification was performed by ESI/MS. Isomeric structures were confirmed by NMR analyses. At the end of the experiment, 12.2% of the administered radioactivity was recovered in bile. As expected, hydroxylation and glucuronidation were the predominant metabolic pathways. The 7-O-glucuronide-fluoranthene metabolite (representing 13.3% of total radioactivity found in bile), 8-O-glucuronide-fluoranthene (11.8%), trans-2,3-dihydro-3-hydroxy-2-O-glucuronide-fluoranthene (17.9%), and cis-2,3-dihydro-2-hydroxy-3-O-glucuronide-fluoranthene (13.9%) were the major metabolites observed in bile. Minor metabolites, such as trans-2,3-dihydro-2-hydroxy-3-O-glucuronide-fluoranthene (3.9%) and 2,3-di-O-glucuronide-fluoranthene (6.6%), also were identified. The 2,3-dihydrodiol-fluoranthene metabolite, which is found in bile conjugated to glucuronic acid, would be, after hydrolysis of the conjugates, a suitable biomarker of PAH pollution in the marine environment.

Comparison of the in vivo biotransformation of two emerging estrogenic contaminants, BP2 and BPS, in zebrafish embryos and adults

Zebrafish embryo assays are increasingly used in the toxicological assessment of endocrine disruptors. Among other advantages, these models are 3R-compliant and are fit for screening purposes. Biotransformation processes are well-recognized as a critical factor influencing toxic response, but major gaps of knowledge exist regarding the characterization of functional metabolic capacities expressed in zebrafish. Comparative metabolic studies between embryos and adults are even scarcer. Using 3H-labeled chemicals, we examined the fate of two estrogenic emerging contaminants, benzophenone-2 (BP2) and bisphenol S (BPS), in 4-day embryos and adult zebrafish. BPS and BP2 were exclusively metabolized through phase II pathways, with no major qualitative difference between larvae and adults except the occurrence of a BP2-di-glucuronide in adults. Quantitatively, the biotransformation of both molecules was more extensive in adults. For BPS, glucuronidation was the predominant pathway in adults and larvae. For BP2, glucuronidation was the major pathway in larvae, but sulfation predominated in adults, with ca. 40% conversion of parent BP2 and an extensive release of several conjugates into water. Further larvae/adults quantitative differences were demonstrated for both molecules, with higher residue concentrations measured in larvae. The study contributes novel data regarding the metabolism of BPS and BP2 in a fish model and shows that phase II conjugation pathways are already functional in 4-dpf-old zebrafish. Comparative analysis of BP2 and BPS metabolic profiles in zebrafish larvae and adults further supports the use of zebrafish embryo as a relevant model in which toxicity and estrogenic activity can be assessed, while taking into account the absorption and fate of tested substances.

Ex vivo gastrointestinal biotransformation of chlorothalonil in the germ-free and conventional rat

1. The metabolism and absorption of chlorothalonil and corresponding diglutathione and dicysteine conjugates was studied using isolated everted gastrointestinal sacs of the conventional and germ-free rat. An HPLC method was used to analyse mucosal and serosal fluids. Thiol metabolites of chlorothalonil were determined by GC/MS. 2. Low absorption of the substrates was observed, with < 4% of the radioactivity being recovered from the serosal buffers and the digestive tissues. A major part of the radioactivity was recovered from the mucosal fluids and it corresponded to unchanged chlorothalonil. Traces of unchanged chlorothalonil and mono-, di- and trimethylthio metabolites were present in serosal fluids as well as unidentified polar peaks. An important transformation (> 75%) of the chlorothalonil conjugates was observed. The di- and trimethylthio metabolites of chlorothalonil were detected from both sides of the everted gut sac of rat incubated with the diglutathione and dicysteine conjugates. 3. Few differences were observed between the conventional and germ-free rat: absorption was higher in the duodenum of germ-free rat, but tissue retention was more significant in the duodenum of the conventional rat.