Bioactivation of quinolines in a recombinant estrogen receptor

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Bioactivation of quinolines in a recombinant estrogen receptor transactivation assay is catalyzed by N-methyltransferases Markus Brinkmann, Bogdan Barz, Danielle Carriere, Mirna Velki, Kilian Smith, Henriette Meyer-Alert, Yvonne Müller, Beat Thalmann, Kerstin Bluhm, Sabrina Schiwy, Simone Hotz, Helena Salowsky, Andreas Tiehm, Markus Hecker, and Henner Hollert Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.8b00372 • Publication Date (Web): 21 Mar 2019 Downloaded from http://pubs.acs.org on March 22, 2019

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Chemical Research in Toxicology

Bioactivation

of

quinolines

in

a

recombinant

estrogen

receptor

transactivation assay is catalyzed by N-methyltransferases Markus Brinkmann1,2* • Bogdan Barz3 • Danielle Carrière1 • Mirna Velki2 • Kilian Smith4 • Henriette Meyer-Alert2 • Yvonne Müller2 • Beat Thalmann2 • Kerstin Bluhm1,2 • Sabrina Schiwy2 • Simone Hotz2 • Helena Salowsky5 • Andreas Tiehm5 • Markus Hecker1 • Henner Hollert2,6,7,8 1School

of Environment & Sustainability and Toxicology Centre, University of Saskatchewan,

Saskatoon, Canada 2Department

of Ecosystem Analysis, Institute for Environmental Research, ABBt – Aachen

Biology and Biotechnology, RWTH Aachen University, Aachen, Germany 3ICS-6: 4Chair

Structural Biochemistry, Forschungszentrum Jülich GmbH, Jülich, Germany

of Environmental Biology and Chemodynamics, Institute for Environmental Research,

ABBt – Aachen Biology and Biotechnology, RWTH Aachen University, Aachen, Germany 5Water 6State

Technology Center, Department of Environmental Biotechnology, Karlsruhe, Germany

Key Laboratory of Pollution Control and Resource Reuse, School of the Environment,

Nanjing University, Nanjing, China 7College 8Key

of Resources and Environmental Science, Chongqing University, Chongqing, China

Laboratory of Yangtze Water Environment, Ministry of Education, Tongji University,

Shanghai, China *Corresponding author: Markus Brinkmann, PhD Phone: +1 (306) 966 1204 E-mail: [email protected]

ACS Paragon Plus Environment

Chemical Research in Toxicology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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TOC/Abstract art:

Abstract: Hydroxylation of polyaromatic compounds through cytochromes P450 (CYPs) is known to result in potentially estrogenic transformation products. Recently, there has been an increasing awareness of the importance of alternative pathways such as aldehyde oxidases (AOX) or N-methyltransferases (NMT) in bioactivation of small molecules, particularly Nheterocycles. Therefore, this study investigated the biotransformation and activity of methylated quinolines, a class of environmentally relevant N-heterocycles that are no native ligands of the estrogen receptor (ER), in the estrogen-responsive cell line ERα CALUX. We found that this widely-used cell line over-expresses AOXs and NMTs while having low expression of CYP enzymes. Exposure of ERα CALUX cells to quinolines resulted in estrogenic effects, which could be mitigated using an inhibitor of AOX/NMTs. No such mitigation occurred after co-exposure to a CYP1A inhibitor. A number of N-methylated but no hydroxylated transformation products were detected using LC-MS, indicating that biotransformations to estrogenic metabolites were likely catalyzed by NMTs. Compared to the natural ER ligand 17β-estradiol, the products formed during the metabolization of quinolines were weak to moderate agonists of the human ERα. Our findings have potential implications for the risk assessment of these compounds and indicate that care must be taken when using in vitro estrogenicity assays, e.g. ERα CALUX, for the characterization of N-heterocycles or environmental samples that may contain them.

Keywords:

ERα CALUX • estrogenicity • hetero-PAHs • NSO-PAC • Methylation • Aldehyde oxidase


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1. Introduction In recent years, mechanistic in vitro bioassays have become increasingly important as tools to identify the molecular and biochemical targets through which chemicals may exert toxic effects in organisms.1-3 Such bioassays may be used to assess mutagenic and non-mutagenic genotoxicity, inhibition or induction of enzymes, and activation or inhibition of nuclear and other receptors.4-6 In particular, the interaction of xenobiotics with hormone receptors, such as the androgen and the estrogen receptor (ER) have received much attention in recent years. Some of these in vitro bioassays have become part of lower tiers of national and international testing programs for endocrine disrupting chemicals (EDCs), i.e. chemicals that may interfere with the hormone system of organisms, such as the Endocrine Disruptor Screening Program of the U.S. Environmental Protection Agency.7-8 In these tiered testing strategies, only chemicals that are suspected to be EDCs based on in vitro assays would progress to higher tiers, e.g. in vivo tests, thereby reducing unnecessary costs and animal experiments.9 Biotransformation of chemicals in these in vitro assays can be a major confounding factor. In many cases, biotransformation leads to detoxification of parent compounds, while in some cases it can also lead to the formation of active metabolites.10 In all instances, the assay would reflect the combined effects of parent and metabolite, rather than those of the parent alone, which is a major challenge in subsequent chemical risk assessments. Two fundamentally different experimental approaches have been followed classically to account for biotransformation. One approach is to use assays that are representative of the biological system of interest, including in vivo metabolic conditions and molecular targets of toxicants.11-12 The second approach involves test systems that lack metabolic enzymes or only express them at very low levels, e.g. recombinant bacteria or yeast.12-13 These systems are comparably artificial and their results do not easily translate to the biological system of interest. However, they facilitate studying the effects of chemicals in absence of their transformation products and leave the option to use exogenous metabolizing systems, e.g. post-mitochondrial supernatant or



microsomal preparations.14 A growing number of recently developed recombinant receptor transactivation assays seek to combine the advantages of both systems in one assay: while they apply human or mammalian cells that express nuclear receptors and the pathways through which they exert biological effects, they make increasing use of cell lines that show only low biotransformation capacity (e.g. the human osteosarcoma cell line U2OS or the African green monkey cell line COS-7), leaving the option to use exogenous activation if required.15 Most commonly, cells with minimal activity of cytochrome P450-dependent monooxygenases (CYPs) are preferred, because CYPs are known to catalyze a wide range of biological oxidation reactions of endogenous and xenobiotic substrates.16 Here, we propose that this strategy may lead to unexpected outcomes that could impede the use of newly developed assays in environmental risk assessments. If optimization is solely focussed on reducing CYP-mediated biotransformation without studying the potential overexpression of other classes of biotransformation enzymes, there is a risk of attributing some of the observed effects to the parent compound that might in fact have been caused by metabolites. The cytosolic aldehyde oxidases (AOXs) and N-methyltransferases (NMTs) can play an important role in the biotransformation of specific groups of chemicals, such as N-heterocyclic pharmaceuticals and xenobiotics, and these processes are not captured when focussing on CYPmediated biotransformation alone.21-22 In this study, we aimed to investigate if bioactivation through AOXs and NMTs is a contributing factor to the observed estrogenicity of selected N-heterocycles in the ERα CALUX, a highly sensitive and popular recombinant human estrogen receptor alpha (ERα) transactivation assay based on the osteosarcoma cell line U2OS.23 Both aox1 and nicotinamide N-methyl transferase (nnmt) genes have been previously shown to be over-expressed in certain types of cancers and cancer cell lines,24-25 thus potentially rendering ERα CALUX cells useful to improve our understanding of the role of AOX and NMTs in the bioactivation of Nheterocycles. We chose methylated quinolines as a class of environmentally relevant model N-


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heterocyclic chemicals, while acknowledging that there is a large variety of other Nheterocycles that should be studied in future research. A great variety of related compounds can be found in ground water at tar oil-contaminated sites and in the blood of tar workers occupationally exposed through inhalation of the tar’s volatile fraction. Some methylated quinolines are also intentionally used as flavors and fragrances.26 In addition to their welldescribed mutagenicity and acute toxicity,27 we recently demonstrated that they may also exhibit estrogenic effects after hydroxylation in another recombinant transactivation assay, rendering them particularly interesting for this study.28 The main aim of the present study was to determine the transactivation potential of methylated quinolines in the ERα CALUX assay, and whether this is due to binding of parent chemicals or transformation products to the estrogen receptor. Here, we found that the observed estrogenicity was mitigated by an inhibitor of AOX and NMTs, respectively, but not CYP1A. Thus, we characterized the ERα CALUX cell line, and compared it with the ER CALUX cell line based on T47Dluc cells, regarding the expression of AOX and NMTs. Last, we quantified the binding affinity of selected methylated and hydroxylated quinolines to the human ERα, both in vitro and in silico, and identified transformation products formed during incubation of quinolines with the cells. It is acknowledged that observations using this widely used bioassay based on a cancer cell line might not be indicative of the relative contribution of CYPs, AOX and NMTs to the biotransformation of N-heterocycles under physiological conditions.

2. Materials and methods 2.1 Chemicals Quinoline (98 %), 2- (97 %), 3- (98 %), 4- (98 %), 6- (98 %), 7- (97 %) and 8-methylquinoline (98 %), 2,6-dimethylquinoline (98 %), 2- (99 %), 6- (96 %) and 8-hydroxyquinoline (99 %), as well as 2-hydroxy-4-methylquinoline (98 %) and 4-hydroxy-2-methylquinoline (98 %) were



obtained from ABCR chemicals (Karlsruhe, Germany). The chemicals 2,4-dimethylquinoline (95 %), 4- (98 %) and 5-hydroxyquinoline (99 %), and 6-hydroxy-2-methylqinoline (98 %) were supplied by Alfa Aesar (Karlsruhe, Germany). Phenanthridine (98 %), 1methylquinolinium iodide (Aldrich CPR), 3-methylisoquinoline (98 %), hydralazine (98 %), αnaphthoflavone (98 %) and 17β-estradiol (E2, 98 %) were purchased from Sigma Aldrich (Steinheim, Germany). Stock solutions of the investigated chemicals were prepared in dimethyl sulfoxide (DMSO, Sigma-Aldrich) and stored at -20°C in the dark until used.

2.2 Radioligand binding assay Competitive binding of the investigated quinolines to human ERα was assessed by SB Drug Discovery (Glasgow, Scotland). Briefly, [3H]-E2 (1 nM final concentration) was mixed with cytosol preparations of Sf-9 cells infected with recombinant baculovirus coding for human ERα in a total volume of 30 µL assay buffer (10 mM Tris, 2 mM DTT, 1 mg mL-1 BSA, 10 % glycerol, pH 7.5). Stock solutions of the investigated quinolines (200 mM) were prepared in DMSO. All subsequent assays were performed in duplicate at a final DMSO concentration of 5 %. 5-Hydroxyquinoline and 2-hydroxy-4-methylquinoline were tested against human ERα at 1 mM starting concentration, while all other compounds were tested at 10 mM starting concentration following a 12-point, half-log serial dilution. Plates were incubated 60 min at room temperature. Bound and free radioligands were separated by the addition of 30 µL of a activated charcoal suspension (2 % charcoal, 0.5 % dextran in 10 mM Tris, 1 mM EDTA, pH 7.5). Plates were centrifuged and 10 µL of the supernatant added to 50 µL of MicroScint (Perkin Elmer, Boston, USA). Plates were read on a Topcount liquid scintillation plate reader (Packard Bioscience, Shelton, USA). A half-log serial dilution series of diethylstilbestrol starting at 10 pM was used as the reference standard. Results were plotted using GraphPad Prism 7.0 and IC50 values interpolated using four parameter logistic regression.


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2.3 In silico analysis of receptor binding We calculated binding affinities for all ligands and E2 using three different methods: "Autodock rigid" (AD), "Autodock flexible" (ADF), and Molecular dynamics (MD) using the linear interaction energy method. Briefly, molecular dynamics (MD) simulations were performed for all ligand-protein conformations docked with the ‘Autodock flexible’ method by use of the software package GROMACS 5.0.5.29 For each receptor-ligand complex we performed 200-ns MD simulations with flexible ligand and protein in explicit water. To estimate the average binding affinities from MD, we selected the lowest affinity interval for each ligand that had the most stable poses (Supporting Information, Section 3.2). Additional details can be found in the Supporting Information.

2.4 ERα CALUX assay U2OS human osteosarcoma cells used in the present study were obtained from and licensed by BioDetection Systems BV (BDS), Amsterdam, Netherlands.23 The assay was performed according to the BDS protocol, with modifications.30-31 Cells were cultured in Dulbecco's modified Eagle's medium (D-MEM)/F12 with GlutaMAX™ and phenol red (Thermo Fisher Scientific, Waltham, USA) that was supplemented with 7.5 % fetal bovine serum (FBS), 1 % MEM nonessential amino acids and 0.2% penicillin/streptomycin solution (5000 U mL-1 each). Cultures were incubated at 37°C (5 % CO2). For the assay, cells were seeded at a density of 10,000 cells per well in D-MEM/F12 with L-glutamine and without phenol red that was supplemented with 5 % stripped FBS, 1 % nonessential amino acids and 0.2 % penicillin/streptomycin solution into 96-well microplates. Plates were incubated for 24 h (37°C, 5 % CO2) before exposure. Cells were dosed with a seven-point 1:2 dilution series of each chemical in triplicates for another 24 h. The DMSO concentration in all exposures was 0.1 %. A maximum concentration of 150 mg L-1 was chosen for all tested chemicals and the MTT cell viability assay was conducted according to the protocol of Sanderson et al.,32 with



modifications, to ensure that the tested concentrations did not cause cytotoxicity. An E2 (Sigma-Aldrich) dilution series ranging from 0.1 to 100 pM and a DMSO solvent control (0.1 %) were included in triplicates on each plate. In addition to testing of individual chemicals, cells were co-exposed to 150 mg L-1 3-methylquinoline and 8-methylquinoline, respectively, and either the AOX/NMT inhibitor hydralazine,33-35 or the CYP1A inhibitor α–naphthoflavone (both 25 µM). After 24 h exposure (37°C, 5 % CO2), the cells were lysed with 30 µL lysis reagent. Luciferase activity in lysates was measured after addition of 100 µL glow mix per well using a GloMax®-96 microplate luminometer (Promega, Madison, USA). Statistical analysis of ERα CALUX data followed the recommendations of Villeneuve at al.36 Mean luminescence values of samples and E2 standards were corrected for the response of solvent controls and normalized to the maximum induction of the E2 standard. Scaled values from triplicate experiments were then plotted against nominal exposure concentrations using GraphPad Prism 7.0 software (GraphPad, San Diego, USA) and fitted using four-parameter logistic regression with variable slope (top and bottom of the curve were set to 1 and 0, respectively). EEF20-80 ranges, i.e. multiple estradiol equivalence factor estimates (EEFs, Equation 1) along the concentration-response curves based on EC20s, EC50s and EC80s, were calculated to both test the assumptions of parallelism and equal efficacy and to provide a measure of uncertainty for mass-balance analyses. Differences between groups exposed to 3methylquinoline and 8-methylquinoline alone were compared to groups that were co-exposed to the AOX/NMT inhibitor hydralazine or the CYP1A inhibitor α–naphthoflavone by means of One-way ANOVA with Dunnett’s multiple comparison test (p ≤ 0.05).

𝐸𝐶𝑋𝐸2

𝐸𝐸𝐹𝑋 = 𝐸𝐶𝑋𝑠𝑎𝑚𝑝𝑙𝑒

(Equation 1)


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2.5 Real-time RT-PCR analyses To characterize ERα CALUX (U2OS) and the closely related ER CALUX cells (based on T47Dluc cells) regarding the importance of AOX- or NMT-mediated bioactivation, we quantified the abundances of transcripts of CYP1A1, aldehyde oxidase 1 (AOX1), nicotinamide N-methyltransferase (NNMT), amine N-methyltransferase (AMT), and phenylethanolamine Nmethyltransferase (PNMT) by means of qPCR. Total RNA was extracted from cell lines using the NucleoSpin RNA Mini kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s protocol. RNA concentration was determined by use of a BioDrop µLITE spectrophotometer (BioDrop, Cambridge, UK). First-strand cDNA was synthesized from 1.25 μg of total RNA (M-MuLV RT, Peqlab, Erlangen, Germany) according to the manufacturer’s protocol. Pre-designed primer pairs for quantitative real-time PCR (qPCR) of abovementioned transcripts were obtained from Sigma Aldrich (Supporting Table S1). Melting curve analyses were performed after real-time PCR to ensure target specificity and single peak amplification. Abundance of mRNA was quantified on a 96-well StepOne Plus real-time PCR system (Applied Biosystems, Foster City, CA, USA) using SYBR Green master mix (Applied Biosystems) as previously reported.37 Abundances of transcripts of target genes were normalized to that of the reference gene β-actin. Data were analyzed using the comparative cycle threshold (ΔΔCt) method and differences of expression in U2OS cells compared to T47D cells using pairwise Student’s t-tests, p ≤ 0.05).38

2.6 Transformation of quinolines during incubation with ERα CALUX cells A bioactivation experiment was performed to determine if potentially estrogenic transformation products were formed during incubation with ERα CALUX cells.28 Cells were grown in 75 cm2 cell culture flasks until 90 % confluent and exposed for 24 h to 10 mg L-1 of each of the quinolines in 15 mL fresh culture medium. The final concentration of DMSO in the exposure medium was 0.1 %. A solvent control was treated as the dilutions without addition of substance.



Following 24 h incubation at 37°C, cells were detached with a cell scraper and homogenized with the cell culture supernatant using an electric disperser. The suspension was then centrifuged at 4°C (4,000 × g) and the supernatant stored at -80°C until analysis. The samples were diluted 1:1 with methanol and the medium components allowed to precipitate for 24 h before transferring an aliquot of the clear supernatant to an HPLC vial with insert. The quinolines were measured using an Agilent 1200 liquid chromatography (LC) system connected to a Thermo Scientific LTQ Orbitrap XL equipped with a heated electrospray ion source run in positive ionization mode. The source temperature was set to 250°C, ion spray voltage to 5 kV, sheath gas to 30 arbitrary units (AU), auxiliary gas to 5 AU, sweep gas switched off and the ion transfer capillary kept at 275°C and at 35 V. Measurement was performed using the linear ion trap of the LTQ Orbitrap XL. Initial testing in MS/MS mode indicated a very low abundance of the parent compound fragments, and the samples were therefore run in single ion monitoring (SIM) mode. Based on the initial testing using pure standards the following [M+H]+ SIM masses were selected: quinoline m/z 130, methylquinolines m/z 144, dimethylquinolines m/z 158, hydroxyquinolines m/z 146 and hydroxymethylquinolines m/z 160. Samples were separated on a Phenomenex Synergi 4µ Fusion-RP 80A HPLC column (250 × 4.6 mm, 4 µm particle size). The injection volume was 10 µL and the column was kept at 22°C. The flow was kept constant at 0.2 mL min-1 and the total run time was 15 min. A solvent gradient of ultrapure water and methanol (both with 0.1% formic acid) was used: 90% water for 1 min, decrease to 5% water over 2 min, held for a further 7 min before returning to the starting conditions of 90% water within 1 min and a final 4 min equilibration period prior to the next injection. Since not all the quinolines could be baseline separated, individual sets of calibration standards (0.003 to 1 mM) were made for each compound. Compounds were identified using the above [M+H]+ SIM masses and retention times as determined from the calibration standards.


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3. Results and Discussion The main aim of the present study was to determine the transactivation potential of various quinolines in the ERα CALUX assay and whether this was due to binding of parent chemicals or transformation products to the ER. We found that inhibitors of AOX and NMTs, respectively, but not of CYP1A could mitigate the observed estrogenicity. Thus, we also characterized the ERα CALUX cell line regarding their expression of AOX and NMTs. Last, we describe the binding affinity of selected methylated and hydroxylated quinolines to the human ERα, both in vitro and in silico, and identified transformation products formed during incubation of quinolines with the cells.

3.1 ERα CALUX transactivation assays The EC50 of E2 in the present study was 6.19 ± 1.98 pM (mean ± standard deviation, n=17), which is within the range reported for the assay in the literature.23, 39 Concentration-response curves were fitted for each of the investigated compounds (Figure 1). Quinoline, 2-, 5-, and 8hydroxyquinoline, 2-methylquinoline, as well as 2-hydroxy-4-methyl- and 4-hydroxy-2methylquinoline were inactive in the ERα CALUX assay, i.e. did not reach an induction of 20 % E2-max. EC20s, EC50s and EC80, as well as corresponding EEFs and EEF20-80 ranges were calculated for all other tested compounds (Table 1). The substances 4- and 6-hydroxyquinoline, 4-, 6-, and 7-methylquinoline, as well as 2,4-dimethyl- and 2,6-dimethlyquinoline did not reach 80 % induction of the E2 standard, making extrapolation beyond the measured range of response necessary to establish the EEF20-80 ranges as recommended by Villeneuve at al.36 EC50s were in the high µg L-1 to low mg L-1 range, and thus EEFs were generally lower compared to those of other non-steroidal EDCs of concern that are commonly found in surface waters, such as bisphenol A, phthalates, select alkylphenols or pesticides.14, 40-41 Environmental levels of methylquinolines in surface waters are often considerably lower (ng L-1 range), but concentrations up to 800 µg L-1 have been measured in groundwater and industrial effluents.42-43



Some of the tested chemicals are used as flavors and fragrances in food products. Maximum use levels of 5 mg kg-1 are reported for “ready-to-eat savories”, and up to 10 mg kg-1 in “bakery wares”.44 Additionally, humans may be exposed during manufacturing processes making use of methylquinolines, including pharmaceuticals, steel, dyes, food coloring, and other organic chemicals.45 They are also present in complex mixtures, including coal tar, byproducts of wood processing, and urban particulate matter. 45 Whiskey, tea and tobacco smoke have been shown to contain considerable levels of methylquinolines.45-46 Although the total human exposure to methylated quinolines does not likely pose a risk to the average population, caution is warranted for occupationally exposed workers.

3.2 Receptor binding affinity and effects of enzyme inhibitors on estrogenicity of quinolines An initial competitive binding study of the investigated chemicals to the human ERα revealed that only 4- and 6-hydroxyquinoline, 2-, 3-, and 8-methylquinoline, as well as 6-hydroxy-2methylquinoline and quinoline exhibited some receptor binding. With IC50s in the range of or extrapolated beyond the high maximum concentrations, however, this binding was likely nonspecific and does not explain observed EC50s in the ERα CALUX (Supporting Table S3). Furthermore, there was no significant correlation between IC50s and EC50s (Pearson’s correlation coefficient r=0.063, p=0.920). Based on our previous study of other heterocycles in a different cell line, we hypothesized that the observed effects might have been caused by transformation products rather than parent chemicals.28 Co-exposure of cells with the CYP1A inhibitor α-naphthoflavone did not result in reduced bioactivity of 3- and 8-methylquinoline (Figure 2, +ANF). However, their effects were significantly suppressed in the presence of hydralazine, which is a known inhibitor of AOX and several NMTs.33-35 The effect of 3-methylquinoline was reduced by 55 % (One-way ANOVA with Dunnett’s multiple comparison test, p=0.0013), and that of 8-methylquinoline by 23 % (p=0.0324). Based on these findings, it is likely that the transformation of methylquinolines to


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bioactive metabolites was catalyzed by either AOXs or NMTs. An initial assessment using a simple derivatization test revealed that all investigated chemicals could potentially be AOX substrates (Supporting Information, Section 2).

3.3 Expression of CYP1A, AOX, and NMTs in ER (T47D) and ERα CALUX (U2OS) cells The importance of AOX- or NMT-mediated bioactivation in ERα CALUX cells was further supported by measurement of the abundance of transcripts of CYP1A1, AOX1, NNMT, AMT, and PNMT relative to the classical ER CALUX cells (Figure 3). There was no difference in the abundance of transcripts of CYP1A between the two cell lines (Student’s t-test, p=0.191); however, basal expression of AMT and PNMT was significantly lower (0.37- and 0.06-fold) in ERα CALUX compared to ER CALUX cells (p=0.0008 and 0.0004). In contrast, the abundance of transcripts of AOX1 and NNMT was significantly greater in ERα CALUX cells compared to ER CALUX cells by a factor of 15.1- and 16,100-fold, respectively (p=0.0001 and a< ]> pyrene in two cell lines from rainbow trout liver. Journal of Biochemical and Molecular Toxicology 2000, 14 (5), 262-276. 12. Boettcher, M.; Grund, S.; Keiter, S.; Kosmehl, T.; Reifferscheid, G.; Seitz, N.; Rocha, P. S.; Hollert, H.; Braunbeck, T., Comparison of in vitro and in situ genotoxicity in the Danube River by means of


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24.

25. 26. 27. 28.

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29. Hess, B.; Kutzner, C.; van der Spoel, D.; Lindahl, E., GROMACS 4: Algorithms for Highly Efficient, Load-Balanced, and Scalable Molecular Simulation. Journal of Chemical Theory and Computation 2008, 4 (3), 435-447. 30. BDS, Analysis of Estrogen Receptor Mediated Luciferase Activity in ER CALUX Cells, P-BDS011(H). 2007. 31. Välitalo, P.; Perkola, N.; Seiler, T.-B.; Sillanpää, M.; Kuckelkorn, J.; Mikola, A.; Hollert, H.; Schultz, E., Estrogenic activity in Finnish municipal wastewater effluents. Water Res. 2016, 88, 740-749. 32. Sanderson, J. T.; Slobbe, L.; Lansbergen, G. W. A.; Safe, S.; van den Berg, M., 2,3,7,8Tetrachlorodibenzo-p-dioxin and Diindolylmethanes Differentially Induce Cytochrome P450 1A1, 1B1, and 19 in H295R Human Adrenocortical Carcinoma Cells. Toxicological Sciences 2001, 61 (1), 40-48. 33. Chavez-Blanco, A.; Perez-Plasencia, C.; Perez-Cardenas, E.; Carrasco-Legleu, C.; Rangel-Lopez, E.; Segura-Pacheco, B.; Taja-Chayeb, L.; Trejo-Becerril, C.; Gonzalez-Fierro, A.; Candelaria, M., Antineoplastic effects of the DNA methylation inhibitor hydralazine and the histone deacetylase inhibitor valproic acid in cancer cell lines. Cancer cell international 2006, 6 (1), 2. 34. Nakamura, K.; OKADA, T.; ISHII, H.; NAKAMURA, K., Differential effects of α-methyldopa, clonidine and hydralazine on norepinephrine and epinephrine synthesizing enzymes in the brainstem nuclei of spontaneously hypertensive rats. The Japanese Journal of Pharmacology 1980, 30 (1), 110. 35. Strelevitz, T. J.; Orozco, C. C.; Obach, R. S., Hydralazine As a Selective Probe Inactivator of Aldehyde Oxidase in Human Hepatocytes: Estimation of the Contribution of Aldehyde Oxidase to Metabolic Clearance. Drug Metab. Disposition 2012, 40 (7), 1441-1448. 36. Villeneuve, D. L.; Blankenship, A. L.; Giesy, J. P., Derivation and application of relative potency estimates based on in vitro bioassay results. Environmental Toxicology and Chemistry 2000, 19 (11), 2835-2843. 37. Brinkmann, M.; Koglin, S.; Eisner, B.; Wiseman, S.; Hecker, M.; Eichbaum, K.; Thalmann, B.; Buchinger, S.; Reifferscheid, G.; Hollert, H., Characterisation of transcriptional responses to dioxins and dioxin-like contaminants in roach (Rutilus rutilus) using whole transcriptome analysis. Science of The Total Environment 2016, 541, 412-423. 38. Livak, K. J.; Schmittgen, T. D., Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method. Methods 2001, 25 (4), 402-408. 39. Kunz, P.; Simon, E.; Creusot, N.; Jayasinghe, B.; Kienle, C.; Maletz, S.; Schifferli, A.; Schonlau, C.; Ait-Aissa, S.; Denslow, N.; Hollert, H.; Werner, I.; Vermeirssen, E., Effect-based tools for monitoring estrogenic mixtures: Evaluation of five in vitro bioassays. Water Res. 2017, 110, 378388. 40. Preuss, T. G.; Gehrhardt, J.; Schirmer, K.; Coors, A.; Rubach, M.; Russ, A.; Jones, P. D.; Giesy, J. P.; Ratte, H. T., Nonylphenol Isomers Differ in Estrogenic Activity. Environmental Science & Technology 2006, 40 (16), 5147-5153. 41. Houtman, C. J.; van Houten, Y. K.; Leonards, P. E. G.; Brouwer, A.; Lamoree, M. H.; Legler, J., Biological Validation of a Sample Preparation Method for ER-CALUX Bioanalysis of Estrogenic Activity in Sediment Using Mixtures of Xeno-Estrogens. Environmental Science & Technology 2006, 40 (7), 2455-2461. 42. Melcer, H.; Steel, P.; Bedford, W. K., Removal of polycyclic aromatic hydrocarbons and heterocyclic nitrogen compounds in a municipal treatment plant. Water Environ. Res. 1995, 67 (6), 926-934. 43. Turney, G. L.; Goerlitz, D. F., Organic contamination of ground water at Gas Works Park, Seattle, Washington. Groundwater Monitoring & Remediation 1990, 10 (3), 187-198.


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44. EFSA CEF Panel, EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids, Scientific Opinion on Flavouring Group Evaluation 24, Revision 2 (FGE.24Rev2): Pyridine, pyrrole, indole and quinoline derivatives from chemical group 28. EFSA Journal 2013, 11 (11), 3453. 45. Haneke, K. E., Methylquinolines - Summary Report of Toxicological Literature. National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA; Contract No. N01-ES-65402 2002. 46. Nishimura, K.; Masuda, M., Minor constituents of Whiskey Fusel oils. J. Food Sci. 1971, 36 (5), 819-822. 47. Perrin, D. D., Dissociation constants of organic bases in aqueous solution. International Union of Pure Applied Chemistry (IUPAC), Commission on Electrochemical Data 1965. 48. Kuş, N.; Sagdinc, S.; Fausto, R., Infrared Spectrum and UV-Induced Photochemistry of MatrixIsolated 5-Hydroxyquinoline. The Journal of Physical Chemistry A 2015, 119 (24), 6296-6308. 49. Pissios, P., Nicotinamide N-Methyltransferase: More Than a Vitamin B3 Clearance Enzyme. Trends in Endocrinology & Metabolism 2017, 28 (5), 340-353. 50. Wenger, D.; Gerecke, A. C.; Heeb, N. V.; Schmid, P.; Hueglin, C.; Naegeli, H.; Zenobi, R., In vitro estrogenicity of ambient particulate matter: contribution of hydroxylated polycyclic aromatic hydrocarbons. Journal of Applied Toxicology 2009, 29 (3), 223-232. 51. Kamiya, M.; Toriba, A.; Onoda, Y.; Kizu, R.; Hayakawa, K., Evaluation of estrogenic activities of hydroxylated polycyclic aromatic hydrocarbons in cigarette smoke condensate. Food and Chemical Toxicology 2005, 43 (7), 1017-1027. 52. Muthumbi, W.; De Boever, P.; Pieters, J. G.; Siciliano, S.; D'Hooge, W.; Verstraete, W., Polycyclic Aromatic Hydrocarbons (PAHs) and Estrogenic Compounds in Experimental Flue Gas Streams. J. Environ. Qual. 2003, 32 (2), 417-422. 53. Machala, M.; Ciganek, M.; Bláha, L.; Minksová, K.; Vondráčk, J., Aryl hydrocarbon receptormediated and estrogenic activities of oxygenated polycyclic aromatic hydrocarbons and azaarenes originally identified in extracts of river sediments. Environmental Toxicology and Chemistry 2001, 20 (12), 2736-2743. 54. Kuch, B.; Kern, F.; Metzger, J.; Trenck, K., Effect-related monitoring: estrogen-like substances in groundwater. Environmental Science and Pollution Research 2010, 17 (2), 250-260.


Chemical Research in Toxicology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

Page 22 of 27

1

Table 1: Estrogenicity of substituted quinolines in the ERα CALUX assay. EC20, 50 and 80 values were interpolated from the combined regression curves of n = 3

2

independent repetitions per substance. Data for 17β-estradiol represent the mean ± standard deviation of 17 individual EC values. n.d.: not determinable. EC20

EC50

EC80

EEF20

EEF50

EEF80

pM

pM

pM

-

-

-

2-Hydroxyquinoline

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

4-Hydroxyquinoline

9.36 108

4.66 109

2.32 1010

2.69 10-9

1.35 10-9

6.75 10-10

5-Hydroxyquinoline

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

6-Hydroxyquinoline

2.38 108

1.26 109

6.70 109

1.03 10-8

4.07 10-9

1.60 10-9

8-Hydroxyquinoline

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

2-Methylquinoline

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

3-Methylquinoline

1.95 107

3.70 107

7.04 107

1.11 10-7

1.46 10-7

1.93 10-7

4-Methylquinoline

9.26 108

1.82 109

3.45 109

2.35 10-9

3.15 10-9

4.22 10-9

6-Methylquinoline

7.15 108

5.12 109

3.66 1010

2.34 10-9

8.36 10-10

2.99 10-10

7-Methylquinoline

8.53 108

1.70 109

3.39 109

2.63 10-9

3.35 10-9

4.27 10-9

8-Methylquinoline

2.88 107

7.90 107

2.17 108

7.95 10-8

6.54 10-8

5.38 10-8

2,4-Dimethylquinoline

6.29 108

1.34 109

2.87 109

4.86 10-9

6.06 10-9

7.57 10-9

2,6-Dimethylquinoline

1.07 109

5.28 109

2.61 109

3.24 10-9

1.74 10-9

9.35 10-10

2-Hydroxy-4-Methylquinoline

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.


n.d.

n.d.

n.d.

n.d.

n.d.

n.d.


3.93 107

9.12 107

2.12 107

5.14 10-8

6.02 10-8

7.06 10-8

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

2.44 ± 0.70

6.19 ± 1.98

15.74 ± 5.66

1.00

1.00

1.00

Substance

Quinoline 17β-Estradiol

3


Page 23 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


4

Figure captions

5

Figure 1: Concentration-response curves in the ERα CALUX assay for all investigated quinolines

6

(closed circles) and the E2 standard curves (open circles) were measured in n=3 independent

7

experiments. Concentration values on the x-axis refer to nominal exposure concentrations. Dots

8

represent mean values from triplicate measurements in one experiment.

9

Figure 2: Effects of 3-methylquinoline (3-MQ) and 8-methylquinoline (8-MQ), respectively, on ERα

10

CALUX cells in the presence and absence of the AOX/NMT inhibitor hydralazine (+HYD) and the

11

CYP1A inhibitor α–naphthoflavone (+ANF). Bars represent the average, and error bars the standard

12

deviation of n = 3 replicate measurements. Asterisks indicate statistically significant differences

13

compared to the effects observed without inhibitors (One-way ANOVA with Dunnett’s multiple

14

comparison test, p ≤ 0.05).

15

Figure 3: Relative abundance of transcripts of β-actin (ACTB), cytochrome P450 1a1 (CYP1A1),

16

aldehyde oxidase 1 (AOX1), nicotinamide N-methyltransferase (NNMT), amine N-methyltransferase

17

(AMT), and phenylethanolamine N-methyltransferase (PNMT) in ER CALUX (T47D) and ERα

18

CALUX (U2OS) cells. Bars represent the average, and error bars the standard deviation of n = 3

19

replicate measurements. Asterisks indicate statistically significant differences of relative expression in

20

U2OS cells compared to T47D cells (pairwise Student’s t-tests, p ≤ 0.05).

21

Figure 4: Interaction poses of three studied chemicals with the LBA of human estrogen receptor α

22

during their lowest binding affinities. a, d, g: Orientation of 17β-estradiol, 3-methylquinoline and 1,3-

23

dimethyl quinolinium in the binding pocket. The helix 12, responsible for opening and closing the

24

binding pocket, is shown in green. The entire protein is shown is colored by secondary structure elements

25

(π-helix - purple, β-sheet - yellow, turn - cyan, coil - white). The molecule is shown in van der Waals

26

representation with atoms colored by their names (Carbon - cyan, Nitrogen - blue, Hydrogen - white,

27

Oxygen - red). b, e, h: Close-up image of the ligands in the binding pocket with some of the residues

28

within 3 Å distance from the ligand shown in licorice and colored by the amino acid type (white -

29

hydrophobic, blue - positively charged, red - negatively charged, green - polar). Hydrogen bonds are

30

shown in magenta dashed lines. c, f, i: Protein - ligand interaction diagram with a distance cut-off of 3

31

Å. The diagram was produced with the Ligand Interaction script in Schroedinger Maestro.



Figure 1 215x296mm (300 x 300 DPI)


Page 24 of 27

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Figure 2 97x81mm (600 x 600 DPI)



Figure 3 116x76mm (600 x 600 DPI)


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Figure 4 271x244mm (300 x 300 DPI)


Bioactivation of quinolines in a recombinant estrogen receptor

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