Letter Cite This: Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX
pubs.acs.org/journal/estlcu
Structure-Dependent in Vitro Metabolism of Alkyl-Substituted Analogues of Triphenyl Phosphate in East Greenland Polar Bears and Ringed Seals Adelle Strobel,†,‡ Robert J. Letcher,*,†,‡ William G. Willmore,‡ Christian Sonne,§ and Rune Dietz§ †
Ecotoxicology and Wildlife Health Division, Environment and Climate Change Canada, National Wildlife Research Centre, Carleton University, Ottawa, ON K1A 0H3, Canada ‡ Department of Biology, Carleton University, Ottawa, ON K1S 5B6, Canada § Department of Bioscience, Arctic Research Centre, Aarhus University, DK-4000 Roskilde, Denmark S Supporting Information *
ABSTRACT: Organophosphate esters (OPEs), used as plasticizers and flame retardants, are major emerging environmental contaminants in the Arctic. OPEs of environmental interest include triphenyl phosphate (TPHP) and a growing array of alkylsubstituted TPHP analogues. Using a microsomal assay of the liver tissue of polar bears and their ringed seal prey, the comparative in vitro metabolism of TPHP was investigated relative to the analogues of isodecyl diphenyl phosphate (IDDPP), (ptert-butylphenyl) diphenyl phosphate (TBPDPP), tris(p-tert-butylphenyl) phosphate (TTBPP), and two tris(isopropylphenyl) phosphate isomers (T2IPPP and T4IPPP). Polar bear metabolism of the p-tert-butylphenyl-substituted OPEs, TBPDPP and TTBPP, had a substantially slower rate and percent metabolic depletion compared to those with TPHP. Isodecyl- and isopropylsubstituted OPEs, IDDPP, T2IPPP, and T4IPPP, were also more slowly depleted by polar bears than TPHP was. TPHP, IDDPP, T2IPPP, TBPDPP, TTBPP, and T4IPPP were all slowly metabolized by ringed seals. Overall, TPHP in vitro metabolism was clearly affected by o- and p-alkyl substitution that generally hindered the depletion rate. This information is important in understanding the accumulation, biotransformation, and magnification of TPHP and other OPEs in top predator polar bears and their ringed seal prey as well as transfer of OPEs between trophic levels, i.e., ringed seal to polar bears.
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adipose, blubber, and liver.7−12 This has been attributed to rapid metabolism of OPEs as has been shown in vitro for rat (Rattus norvegicus), human (Homo sapiens), herring gull (Larus argentatus), polar bear, and ringed seal in biotransformation assays using liver microsomes.12−15 OP triesters with shorter chain linear alkyl groups and/or less branched alkyl groups are more rapidly metabolized by polar bear and herring gull liver microsomes than are OP triesters with longer chain alkyl groups or aryl groups such as tri(n-butyl) phosphate (TNBP) and tris(2-butoxyethyl) phosphate (TBOEP), tris(2-ethylhexyl)
INTRODUCTION
Organophosphate esters (OPEs) are chemical additives and are used as plasticizers and flame retardants, and as a consequence, OPEs are emerging contaminants that are being increasingly reported in environmental compartments on a global scale. OPEs are present in the Arctic atmosphere, sediment, and seawater and at concentrations that are greater than those of the phased-out and increasingly regulated polybrominated diphenyl ether (PBDE) flame retardants.1−4 OPE exposure for Arctic biota is a concern in addition to there being a dearth of available toxicological data.5,6 From the few monitoring studies that exist, top Arctic predators, including polar bears (Ursus maritimus) and their main prey source, ringed seal (Pusa hispida), have consistently shown low concentrations of OPEs in various storage tissues, including © XXXX American Chemical Society
Received: Revised: Accepted: Published: A
February 6, 2018 February 25, 2018 March 1, 2018 March 1, 2018 DOI: 10.1021/acs.estlett.8b00064 Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX
Letter
Environmental Science & Technology Letters phosphate (TEHP), or triphenyl phosphate (TPHP).12,14 An array of alkyl-substituted TPHPs are emerging environmental contaminants and appear to have been used commercially in Canada as early as 1991 and in the United States as early as 1975.16,17 Alkyl-substituted TPHPs include isodecyl diphenyl phosphate (IDDPP), (p-tert-butylphenyl) diphenyl phosphate (TBPDPP), tris(p-tert-butylphenyl) phosphate (TTBPP), and tris(isopropylphenyl) phosphate isomers (e.g., T2IPPP and T4IPPP). To the best of our knowledge, for any environmental biota or wildlife species, there have been no reports that have examined the biotransformation, metabolism, and fate of triphenyl phosphate (TPHP) as compared to alkyl-substituted TPHP analogues. Alkyl substitution on the three aryl groups of TPHP increases the molecular bulkiness of the TPHP base structure, which could influence biotransformation and half-lives and thus influence the environmental persistence, fate, and bioaccumulation in OPE-exposed biota, including Arctic wildlife. Recently, the long-range transport of OPEs has been assessed using the Organisation for Economic Co-operation and Development (OECD) Screening Tool, which found that IDDPP and TTBPP had high environmental persistence estimates.18 OPEs partition to the particle phase in air, but recent modeling has demonstrated IDDPP can be distributed in both the aerosol and gas phases.19 Although TTBPP has not been reported in Arctic air samples,1 modeling of TTBPP and TIPPP isomers has shown transport distances of 2853 and 2739 km and persistence in air (POV) of 8160 and 2724 h, respectively.1 Additionally, the soil half-life of TTBPP was reported to be 12 h based on estimates using the environmental model CATALOGIC. There are concerns about the potential for these emerging alkyl-TPHP OPEs to have properties of environmental persistence similar to those of PBDEs.19 There is potential for transport and, ultimately, exposure of the more hydrocarbon-like alkyl-substituted TPHPs to Arctic wildlife. This study investigated the effect of differences in the molecular structure of TPHP and several commercially important alkyl-substituted TPHP analogues on in vitro metabolism, using enzymatically viable liver tissue of polar bear compared to that of ringed seal collected from East Greenland in the Arctic.
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Figure 1. Time-course depletion as a result of in vitro assay incubation of organophosphate (OP) triesters in ringed seal (RS) and polar bear (PB) liver microsomes. Compounds triphenyl phosphate (TPHP), isodecyl diphenyl phosphate (IDDPP), (p-tert-butylphenyl) diphenyl phosphate (TBPDPP), tris(p-tert-butylphenyl) phosphate (TTBPP), tris(p-isopropylphenyl) phosphate (T4IPPP), and tris(o-isopropylphenyl) phosphate (T2IPPP) were studied. Time-dependent depletion for PBs is indicated by solid symbols and lines, and RSs are shown with clear symbols dotted lines. Each data point is the mean of three replicate assays conducted on two different days (total of six replicates), and the error bars represent the ±standard error of the mean (SEM). Error bars are not shown where they are smaller than the size of the symbol. Asterisks indicate time points at which OP triester depletion differs significantly (p < 0.01) from the concentration at 1 min.
MATERIALS AND METHODS
Chemicals and Reagents. OP triester and OP diester standard solutions were prepared in methanol. IDDPP (Santicizer-148 Tech Mix including TPHP) and TBPDPP (Tech Mix) were purchased from Chromatographic Specialties Inc. (Brockville, ON). The purities of IDDPP and TBPDPP in these technical mixtures were not available from the suppliers. T4IPPP (>98% purity) and T2IPPP (96% purity) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). TTBPP (purity unknown) and DPHP were purchased from Sigma-Aldrich (Oakville, ON). Mass-labeled internal standards d33-T4IPP (>97% purity) and d21-TCrP (>99% purity) were purchased from Caledon (Georgetown, ON). d10DPHP (>97%) was purchased from Dr. Belov at the Max Planck Institute for Biophysical Chemistry (Gö ttingen, Germany). The chemical structures for the OP triesters under study are shown in Figure 1. Wistar-Han rat liver microsomes (20 mg of microsomal protein/mL) and the nicotinamide adenine dinucleotide phosphate (NADPH) regenerating system (solutions A and B) were purchased
from Corning (Corning, NY). Potassium phosphate buffer (pH 7.4) was purchased from Biotage (Charlotte, NC). Sampling and Tissue Preparation. Polar bear (PB; n = 6) and ringed seal (RS; n = 7) liver tissues were collected from animals harvested by Inuit hunters at Scoresby Sound, Eastern Greenland (70−71°N, 20−21°W) in February and March 2011 and 2012 and frozen at −80 °C within 1 h of being harvested. All sample and sample processing details are fully described by Strobel et al.12 Briefly, liver tissue was differentially centrifuged to separate the microsomal component and pooled (PB, 53.50 mL; RS, 30.65 mL) to provide a sufficient quantity for all in vitro assay experiments for this study. Enzyme viability was determined using the ethoxyresorufin-O-deethylase (EROD) assay to measure the CYP1A monooxygenase-mediated activity.20 The microsomal processing, protein concentrations, and CYP1A-associated enzyme activity have been reported previously and are also listed in Table S1.12 In Vitro Metabolism Assay for OP Triester Depletion and OP Diester Formation. The in vitro metabolism assay for B
DOI: 10.1021/acs.estlett.8b00064 Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX
Letter
Environmental Science & Technology Letters
Table 1. Following a 100 min Incubation of the in Vitro Assay Using Polar Bear and Ringed Seal Liver Microsomes, Mean Percent Depletions [±standard error mean (SEM)] of Organophosphate (OP) Triesters and Mean Percent Conversions (± SEM) of the Parent OP Triester to the Corresponding OP Diester Metabolitea % depletionb (±SEM) OP triester
administered concentration (μM)
TPHPd IDDPP TTBPP TBPDPP T4IPPP T2IPPP
2 2 2 2 2 2
polar bear 99.5 81 67 58 73 83
± ± ± ± ± ±
0.25 4.7 6.6 8.4 5.0 4.3
% conversion to DPHPc (±SEM)
ringed seal
polar bear
ringed seal
± ± ± ± ± ±
97 ± 11 4 ± 0.45 N/D 41 ± 9.8 N/D N/D
60 ± 13 7 ± 5.9 N/D 41 ± 28 N/D N/D
16 44 48 25 62 86
7.1 8.9 7.1 6.8 6.4 2.6
a
The triesters presented are (p-tert-butylphenyl) diphenyl phosphate (TBPDPP), tris(p-tert-butylphenyl) phosphate (TTBPP), tris(pisopropylphenyl) phosphate (T4IPPP), and tris(o-isopropylphenyl) (T2IPPP). Additionally, for IDDPP and TBPDPP, diphenyl phosphate (DPHP) could be quantified. See Table S3 for details regarding nanomole depletion of OP triesters and formation of DPHP. b% depletion = [(administered concentration − OP triester concentration at 100 min)/(administered concentration)] × 100. cWhere applicable, the mean % conversion = [(OP diester formed)/(OP triester depleted)] × 100% ± SEM. dTPHP results reproduced from ref 12.
mm (length) × 2.1 mm (inside diameter), 1.7 μm particle size]. The column temperature was set to 40 °C, and the samples were maintained at 20 °C. The two mobile phases used were methanol and water, each with 2 mM ammonium acetate. The total elution time was 12 min and followed a gradient where the mobile phases started with a flow rate of 0.5 mL/min. The elution gradient was as follows: initially 5% methanol with 2 mM ammonium acetate increasing to 95% within 5 min where it was held for 2 min. The initial conditions of the 5% methanol with 2 μM ammonium acetate were then maintained for an additional 5 min. The metabolite DPHP was analyzed by UPLC−MS/MS in the ESI(−) mode for samples dosed with IDDPP, and the results were compared to the results from the ESI(+) mode. There was little difference in the results, and thus, ESI(−)based quantification was used. However, TBPDPP-dosed samples were measured using ESI(+) as the additional ESI(−) analysis was deemed unnecessary. ESI(+)-MS/MS analysis of the OPEs was based on multiplereaction monitoring (MRM) ion channels and UPLC retention times compared to authentic OPE standards or in technical mixtures, and their characteristic mass transitions were observed by MRM (Table S2). Integration of chromatographic peaks generated by UPLC−ESI-MS/MS analysis was automatic, and quantification was based on the internal standard isotope dilution approach. Calibration curves based on five- to seven-point OPE concentrations were analyzed by UPLC−ESIMS/MS with each batch of in vitro assay samples and were highly linear with r2 consistently well above 0.90 (Figure S1). OPE quantification was accomplished using MassLynx version 4.1. Data Analysis and Statistics. Time-course regressions from the OPE in vitro assays were plotted using GraphPad Prism version 7.0c (GraphPad Software, San Diego, CA). Regressions used the one-phase decay (polar bear degradation of IDDPP), one-phase association (DPHP), or linear regressions (all other OP triesters presented) and least-squares fit functions. The nonparametric Friedman test paired with Dunnett’s multiple-comparison test was conducted to determine the level of significance (p < 0.01) at each time point compared to the initial concentration measured at 1 min.
OP triesters was conducted according to our previously published method12 as well as based on methods reported elsewhere.12,14,15 Briefly, liver microsomes were administered a 2 μM concentration of a given OP triester solution. We showed previously that in herring gull liver microsomal assays at similar protein concentrations, 2 μM was found to be ≫2KM; thus, the enzymes are substrate-saturated, and enzyme kinetics are zeroorder.14 The solution was incubated at 37 °C with an 80 rpm shaker in the presence of the NADPH regenerating system. Aliquots of 100 μL of the incubation solution were taken at 1, 2, 5, 10, 40, 70, and 100 min with each being added to the methanol-containing internal standard mixture (25 ppb). The solution was microcentrifuged at 10000 rpm (6708g) for 5 min (Eppendorf Mini Spin Plus with a model F-45-12-11 rotor). The resulting solutions were diluted to ensure that the analyte concentrations were within the linear response range in the subsequent ultra-high-pressure liquid chromatography (UPLC)−mass spectrometry (MS) analysis.12 TTBPP aliquots required additional dilution (i.e., 100 μL of sample and 100 μL of MeOH) that was also necessary for T4IPPP and T2IPPP (i.e., 100 μL of sample and 500 μL of methanol). The quality control included intraday triplicates, blanks, and negative controls based on deactivated Wistar-Han rat liver microsomes. A negative control was also included in which the in vitro assay for polar bear microsomes lacked NADPH. Metabolic depletion of TPHP was shown to occur in the absence of NADPH. The TPHP to DPHP conversion was on the order of 70% for the negative control assay without NADPH, whereas the same assay in the presence of NADPH resulted in a TPHP to DPHP conversion of 23%. NADPH-independent TPHP metabolism could be a substantial contribution to TPHP and alkyl-TPHP metabolism in polar bear. OP Triester and Diester Determination from the in Vitro Biotransformation Assay. Quantification of OP triesters and their diester metabolites was conducted by utilizing a Waters Acquity ultra-high-pressure liquid chromatography (UPLC) instrument coupled to a Waters Xevo TQMS system operated in the electron spray ionization (ESI)+ mode. The approach was the same as for OPE analysis conducted previously.12,21−23 Briefly, the injection volume was 10 μL. The ESI(+) mode conditions were as follows: 1.0 kV capillary voltage, 150 and 600 °C source and desolvation temperatures, respectively, and 800 and 150 L/h desolvation gas and cone gas flow rates, respectively. Analytes were separated using a Waters Aquity UPLC BEH C18 column [50
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RESULTS AND DISCUSSION The in vitro metabolism of TPHP in polar bear microsomal assays was rapid, with complete metabolic depletion having C
DOI: 10.1021/acs.estlett.8b00064 Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX
Letter
Environmental Science & Technology Letters
rings (e.g., T2IPPP) appeared to promote in vitro metabolism, as compared to the almost nonexistent metabolism for TPHP. OP diester metabolite formation was a small contributor to the OP triester depletion mass balance, where DPHP could be measured, as in the case of IDDPP and TBPDPP (Figure S2 and Table 1).12 That is, DPHP contributed
