Characterized in Vitro Metabolism Kinetics of Alkyl Organophosphate

Feb 13, 2018 - Key Laboratory of Drinking Water Science and Technology, Research ... fish samples collected from European countries,(5-8) the Philippi...
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Article Cite This: Environ. Sci. Technol. 2018, 52, 3202−3210

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Characterized in Vitro Metabolism Kinetics of Alkyl Organophosphate Esters in Fish Liver and Intestinal Microsomes Rui Hou,†,§ Chao Huang,†,§ Kaifeng Rao,‡ Yiping Xu,*,† and Zijian Wang‡ †

Key Laboratory of Drinking Water Science and Technology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China ‡ State Key Laboratory of Environmental Aquatic Chemistry, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China § University of Chinese Academy of Sciences, Beijing 100049, China S Supporting Information *

ABSTRACT: Tris(2-butoxyethyl) phosphate (TBOEP) and tris(n-butyl) phosphate (TNBP) are the most commonly used alkyl organophosphate esters (alkyl-OPEs), and they increasingly accumulate in organisms and create potential health hazards. This study examined the metabolism of TNBP and TBOEP in Carassius carassius liver and intestinal microsomes and the production of their corresponding monohydroxylated and dealkylated metabolites. After 140 min of incubation with fish liver microsomes, the rapid depletion of TNBP and TBOEP were both best fitted to the Michaelis−Menten model (at administrated concentrations ranging from 0.5 to 200 μM), with a CLint (intrinsic clearance) of 3.1 and 3.9 μL·min−1·mg−1 protein, respectively. But no significant (P > 0.05) biotransformation was observed for these compounds in intestinal microsomes at any administrated concentrations. In fish liver microsomes assay, bis(2-butoxyethyl) hydroxyethyl phosphate (BBOEHEP) and bis(2-butoxyethyl) 3-hydroxyl-2-butoxyethyl phosphate (3-OH-TBOEP) were the most abundant metabolites of TBOEP, and dibutyl-3-hydroxybutyl phosphate (3-OH-TNBP) was the predominant metabolite of TNBP. Similarly, the apparent Vmax values (maximum metabolic rate) of BBOEHEP and 3-OH-TNBP were also respectively highest among those of other metabolites. Further inhibition studies were conducted to identify the specific cytochrome P450 (CYP450) isozymes involved in the metabolism of TNBP and TBOEP in liver microsomes. It was confirmed that CYP3A4 and CYP1A were the significant CYP450 isoforms catalyzing the metabolism of TNBP and TBOEP in fish liver microsomes. Overall, this study emphasized the importance of hydroxylated metabolites as biomarkers for alkyl-OPEs exposure, and further research is needed to validate the in vivo formation and toxicological implications of these metabolites.

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fish samples collected from European countries,5−8 the Philippines,9 Canada,10 and China11,12 with total levels ranging from 1.43 to 6000 ng·g−1 lipid weight (lw). A recent study also reported the biomagnification of TBOEP in the benthic and pelagic food web and its accumulation in predatory fish from the Netherlands.5 Very recently, we demonstrated TNBP was the most accmulated OPE in the liver of freshwater fish from Beijing, China, and proposed a preliminary interpretation for the relationship between tissue distribution and the metabolism process in fish.11 Several toxicological studies have also demonstrated that TBOEP and TNBP could display various adverse effects to animals and human health.3,13 TBOEP can induce oxidative

INTRODUCTION Organophosphate esters (OPEs) have been used as flame retardants and plasticizers for decades in a variety of industrial and consumer products, including textiles, plastics, electronics, upholstered furniture, and baby products.1 Alkyl-substituted organophosphate esters (alkyl-OPEs) are a major group of OPEs that includes tris(2-butoxyethyl) phosphate (TBOEP), tris(n-butyl) phosphate (TNBP), tris(ethyl) phosphate (TEP), and tris(2-ethylhexyl) phosphate (TEHP). The global production of TBOEP and TNBP has been estimated at 5000−6000 tons and 3000−5000 tons per year, respectively.2,3 TBOEP and TNBP are predominantly used as plasticizers, lubricants, and flame retardants.1 Due to their vast usage, alkyl-OPEs can be ubiquitously distributed in indoor dust, the atmosphere, soil, surface water, and sediment and accumulate to high levels in animals, especially for aquatic organisms.4 TBOEP and TNBP were the major OPEs among those monitored in marine and freshwater © 2018 American Chemical Society

Received: Revised: Accepted: Published: 3202

November 14, 2017 January 18, 2018 February 13, 2018 February 13, 2018 DOI: 10.1021/acs.est.7b05825 Environ. Sci. Technol. 2018, 52, 3202−3210

Article

Environmental Science & Technology

dithiothreitol, NADPH, ketoconazole (KTZ), α-naphthoflavone (ANF), diethyldithiocarbamate (DDC), and quinidine (QUI) were all obtained from Sigma-Aldrich (St. Louis, MO, USA). All organic solvents used in this study were HPLC grade. In Vitro Metabolism Assay. Adult crucian carps, approximately 20 ± 6 cm in length and 212 ± 15 g in weight (n = 5), were purchased from a local market and maintained with filtered dechlorinated water in the laboratory (at 25 ± 2 °C) for 2 weeks prior to sacrifice. The preparation methods for liver and intestinal microsomes were adopted from Stapleton et al.31 and Lindström-Seppä et al.32 respectively, and the detailed procedure is available in the Supporting Information (section S1). The total protein concentrations of microsomes were determined using the bicinchoninic acid (BCA) assay kit (Biomiga, Shanghai, China). The CYP1A enzyme activity was determined using the ethoxyresorufin O-deethylase (EROD) assay as described by Kennedy and Jones,33 while the CYP 3A4 enzyme activity was quantified using 7-benzyloxy-4-(trifluoromethyl) coumarin (BFCD) according to the method of Li et al.34 The mean EROD activity in liver and intestinal microsomes was 53.3 ± 2 and 4.2 ± 0.4 pmol·mg−1·min−1 protein, respectively, and the BFCD activity in liver and intestinal microsomes was 3.9 ± 1.1 and 0.3 ± 0.1 pmol·mg−1· min−1 protein, respectively. These activity levels were comparable to those of other fish microsome preparations,32,35,36 which confirmed the activity and suitability of the processed microsomes for studying the in vitro metabolism of the two alkyl-OPEs. The in vitro incubations were conducted in glass tubes according to previously described procedures.35,37,38 The incubation mixtures consisted of 3 μL of each OPE (TBOEP and TNBP) in dimethyl sulfoxide (DMSO) (final administered concentration range of 0.5 to 200 μM), 477 μL of incubation buffer (final concentrations of 0.1 M NaH2PO4, 1 mM EDTA, 10 mM dithiothreitol, and 100 μM NADPH buffer; pH = 7.4), and 20 μL of microsomes (final concentration of 1 mg·mL−1 protein) in a total volume of 500 μL. The metabolism was initiated by adding NADPH buffer, and reactions were incubated at 25 °C for 0, 2, 4, 10, 20, 80, 140, or 200 min. Procedural blank samples consisted of the incubation buffer and microsomes only. Negative control samples were prepared as described above but with the heat-inactivated microsomes (10 min at 100 °C) at all administered concentrations for the two OPEs. At the appropriate time interval, the incubation was stopped by the addition 500 μL of TBOEP-d6, TNBP-d27, and BBOEP-d4 in ice cold methanol (20 ng for each internal standard) and extracted according to a previously reported method.28 Briefly, samples were vortexed for 30 s, ultrasonicated for 5 min, and centrifuged at 3500g for 5 min. The supernatant was collected, and the liquid−liquid extraction procedure was repeated twice. The combined extracts were evaporated under a gentle stream of nitrogen to 500 μL, then filtered through a 0.2 μm filter (GHP Acrodisc; PALL, Dreieich, Germany), and immediately stored at −20 °C until analysis. Each experimental condition was repeated three times independently over the course of a week to ensure its reproducibility. Enzymes Inhibition Assay. An inhibition study of the metabolism of TNBP and TBOEP in liver microsomes was performed according to the method of Shen et al.36 Ketoconazole (KTZ), α-naphthoflavone (ANF), quinidine (QUI), and diethyldithiocarbamate (DDC) are commonly used inhibitors to CYP3A4, CYP1A, CYP2D6, and CYP2E1,

stress in mouse TM3 Leydig cells and increase 17-estradiol (E2) and testosterone (T) concentrations in human H295R cells.13,14 TBOEP can disturb the endocrine axes, including the hypothalamus-pituitary-thyroidal, hypothalamus-pituitaryadrenal, and hypothalamus-pituitary-gonadal axes,15 and interfere with the neurodevelopment of zebrafish larvae.16 TBOEP and TNBP can also reduce the levels of antioxidant enzymes and heat shock protein related genes in Asian freshwater clams.17 Numerous in vivo and in vitro studies have shown that OPEs can be rapidly transformed to metabolites through the phase I metabolic reactions (i.e., dealkylation and hydroxylation) and phase II conjunction reactions.18−24 Dialkyl phosphates (DAPs) resulting from dealkylation metabolism, such as di(nbutyl) phosphate (DNBP), bis(2-butoxyethyl) phosphate (BBOEP), diphenyl phosphate (DPHP), and bis(1,3-dichloro2-propyl) phosphate (BDCIPP), have been characterized as the major metabolites in recent human and animal biomonitoring studies.11,24−27 However, some new evidence suggests that the oxidative metabolites (OH-OPEs) resulting from hydroxylation are more significant metabolites than DAPs for individual OPEs. Van den Eede et al. reported that bis(2-butoxyethyl) hydroxyl-3-butoxyethyl phosphate (3-OH-TBOEP) and bis(2butoxyethyl) hydroxyethyl phosphate (BBOEHEP), rather than BBOEP, were the major metabolites of TBOEP in human liver microsomes.28 However, to our knowledge no characterization is available for the metabolites and pathways involved in alkylOPE metabolism in fish. Investigating the metabolism kinetics of TNBP and TBOEP and the in vitro formation of their metabolites by fish liver microsomes would contribute significantly to a better understanding of alkyl-OPEs metabolic pathways in fish. Cytochromes P450 (CYPs) are the major enzyme systems involved in the hepatic metabolism of OPEs.18,23,29 However, specific CYP enzymes involved in the biotransformation of OPEs have not been identified. In addition, although the intestine is another site of CYP, especially CYP3A,30 expression in fish, its contribution to the metabolism of OPEs has not been investigated. Therefore, our primary purpose was to compare the metabolic kinetics of TNBP and TBOEP in liver and intestinal microsomes from crucian carp (Carassius carassius). In particular, the maximum velocity (Vmax) and Michaelis constant (Km) associated with the depletion of TNBP and TBOEP were determined. The formation rates of BBEOHEP, 3-OH-TBOEP, and dibutyl-3-hydroxybutyl phosphate (3-OH-TNBP) were evaluated and compared to rates for the former confirmed DAP metabolites, BBOEP and DNBP, in liver microsomes. Finally, the specific CYP isozymes responsible for the metabolism of the alkyl-OPEs and the production of their metabolites were identified.

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EXPERIMENTAL SECTION Chemicals and Reagents. Standards of TNBP (99% of purity) and TBOEP (99.5% of purity) were purchased from Accustandard, Inc. (New Haven, CT, USA), and the metabolites BBOEP (96% of purity), BBOEHEP (97% of purity), 3-OH-TBOEP (95% of purity), DNBP (98% of purity), and 3-OH-TNBP (96% of purity) were purchased from Toronto Research Chemicals (Toronto, ON, Canada). Internal standards (TBOEP-d6, TNBP-d27, and BBOEP-d4) used in this study were also purchased from Toronto Research Chemicals (Toronto, ON, Canada). NaH2PO4, EDTA, 3203

DOI: 10.1021/acs.est.7b05825 Environ. Sci. Technol. 2018, 52, 3202−3210

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Environmental Science & Technology Table 1. Detailed Information of MRM Transitions Using a Waters ACQUITY UPLC System Coupled to TQ Mass Spectrometer and Quality Control of the Quantified Compoundsa compounds ESI+ TBOEP TBOEP-d6 BBOEHEP 3-OH-TBOEP TNBP TNBP-d27 3-OH-TNBP ESI− BBOEP BBOEP-d4 DNBP a

MRM transitions 399 399 405 405 343 343 415 415 267 267 294 294 283 283

> > > > > > > > > > > > > >

199* 299 202* 303 244* 199 199* 244 99* 211 102* 230 99* 211

297 297 301 301 209 209

> > > > > >

79* 197 79* 199 153* 79

cone (V)

collision energy (V)

retention time (min)

MLOD (ng/mL)

procedure blank (ng/mL)

recovery (%)

precision (% RSD)

30

15

4.22

0.3

0.1

93−98

6.9

30

15

4.23

0.3

0.1

90−101

3.3

25

10

6.91

1.5

0.05). Additionally, KTZ at 50 μM was the only inhibitor with a significant effect on the production of metabolites of BBOEHEP, the sum of all OHTBOEP isomers, DNBP, and 3-OH-TNBP by fish liver microsomes (P < 0.05) (Figure 3). The effects of KTZ, ANF, QUI, and DDC on activities of four CYP isoforms (CYP 1A, 3A4, 2D6, and 2E1) in a parallel panel of fish liver microsomes were also investigated in this study (Figure S4). Because QUI selectively inhibited CYP2D6 activity, our findings suggested that CYP2D6 may contribute to biotransformation of TBOEP and TNBP to some extent. Additionally, the reduced metabolic rates of TBOEP and TNBP were found to be relatively consistent with decreasing microsomal CYP1A activity caused by ANF at the 50 μM concentration. This finding suggested that CYP1A probably plays an important role in metabolism of TNBP and TBOEP. KTZ is commonly used as a specific inhibitor of CYP3A4 in vitro.49 In this study, except for CYP3A4, KTZ also significantly inhibited CYP1A and CYP2D6 activities in fish at the high concentration (50 μM; Figure S4). Therefore, it might cause overinterpretation of inhibition results of the present study. In our view, considering KTZ also vastly inhibited the metabolism of TNBP and TBOEP even at a low concentration (5 μM) when only CYP3A4 activity was significantly decreased, CYP3A4 should play an important role in metabolism of the two alkyl-OPEs and in formation of the hydroxylated and DAPs metabolites. In previous studies, CYP3A4 was also observed for involvement in the structure-similar metabolism of organophosphorus pesticides (chlorpyrifos,50 parathion,51 and diazinon51), polybrominated diphenyl ethers (BDE 99 and BDE 209),46 and tetrabromobisphenol A.36 It is important to note that previous studies found that KTZ can also inhibit other CYP enzymes, such as CYP2A6, CYP2B6, CYP2C8, and CYP2E1, at relatively higher concentrations (>5 μM).49 Feo et al. found CYP2B6 effectively catalyzed in vitro metabolism of BDE 47 and in the formation of hydroxylated metabolites in human liver microsomes.52 The role of other CYP isoforms involved in the alkyl-OPEs metabolism should also be considered further. Implications for in Vivo Toxicokinetics Studies. Our results showed that the metabolism of alkyl-OPEs in fish liver was mediated by CYP1A and CYP3A4. Our analysis also revealed that OPEs were minimally metabolized in the fish intestine, possibly due to the low CYP enzyme activity. Therefore, this study suggests that the fish intestine, although a primary tissue involved in the ingestion of OPEs, will make a

Figure 3. Effects of CYP enzymes inhibitors on metabolism rates of parent compounds depletion and metabolites formation in 200 min incubations of TBOEP (A) and TNBP (B) with fish liver microsomes. Data are means ± SD (n = 3). The asterisks (*) indicate significant difference of metabolism rates under inhibition compared to the control (ANOVA, P < 0.05). Abbreviation: KTZ, ketoconazole; ANF, a-naphthoflavone; QUI, quinidine; DDC, diethyldithiocarbamate.

at both concentrations significantly (P < 0.05) reduced the metabolic rates of both TNBP and TBOEP by more than 63%. Both ANF and QUI at 50 μM significantly reduced the metabolism of TBOEP, whereas only 50 μM ANF inhibited TNBP metabolism significantly (P < 0.05). QUI at 50 μM also inhibited approximately 35% of the metabolic rates of TNBP, 3207

DOI: 10.1021/acs.est.7b05825 Environ. Sci. Technol. 2018, 52, 3202−3210

Environmental Science & Technology limited contribution to the “first-pass” in vivo metabolism of alkyl-OPEs and can thus be excluded from the metabolic compartment in physiologically based pharmacokinetic (PBPK) models. Notably, this finding cannot be extended to aryl-OPEs. TPHP, one of the aryl-OPEs, can be hydrolyzed by serum hydrolases53 and thus may exhibit metabolism kinetics in the intestine that are distinct from those of alkyl-OPEs. We first identified and accurately quantified the metabolites 3-OH-TBOEP, BBOEHEP, and 3-OH-TNBP formed in a fish metabolism study. The significance of these metabolites in this study is in good agreement with human urine monitoring and in vivo rat exposure studies. BBOEHEP was detected as a target biomarker of TBOEP exposure in the majority of urine samples from a group of Australians27 and adults from California,54 and 3-OH-TBOEP was also detected at the same levels as BBOEHEP in human urine collected from subjects from Belgium.55 In the rat exposure study, the OH-TNBP was detected as major metabolites in excreted urine.56 It should also be emphasized that the mono-OH-OPEs metabolites can undergo next phase II conjunction in the absence of glucuronyl transferases, and BBOEHEP could be metabolized through O-dealkylation to BBOEP, according to previous in vitro studies.23,46,47 A recent in vivo fish exposure study qualitatively demonstrated that DAPs accounted for more than 84% of the peak area of TBOEP and TNBP metabolites in the liver of zebrafish, while the BBOEHEP, OH-TNBP, and OH-TBOEP has not been detected.45 The lower contribution of hydroxylation metabolites may be attributed to the rapid glucuronic acid conjugation or other phase II reaction on OHOPEs occurring in the body.45 Kojima et al. reported that DNBP did not exhibit any nuclear receptor activity, although the parent TNBP showed androgen receptor (AR) and glucocorticoid receptor (GR) antagonistic activity; BBOEHEP and 3-OH-TBOEP also acted as pregnane X receptor (PXR) agonists with similar levels to TBOEP, while BBOEP did not show any activity.57 Therefore, because the accumulation of the primary metabolites of OPEs in the body is definitely worthwhile to understand its toxicology to organisms, further in vitro−in vivo extrapolation and in vivo validations studies will be necessary in future.

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ACKNOWLEDGMENTS

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REFERENCES

The research leading to these results was performed with financial support from the National Natural Science Foundation of China (Nos. 21437006, 41571469), the Chinese Academy of Sciences STS Program (KFJ-SW-STS-171), and National Science and Technology Major Project of China (2014ZX07206-005-002).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.7b05825. Details of preparation of liver and intestinal microsomes, OH-TBOEP isomers identification, selection of proper metabolism kinetic models, and depletion kinetics of TBOEP and TNBP (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: +86 (10) 6291 6051; fax: +86 (10) 6292 3541; e-mail: [email protected]. ORCID

Rui Hou: 0000-0002-9614-883X Yiping Xu: 0000-0002-0681-473X Notes

The authors declare no competing financial interest. 3208

DOI: 10.1021/acs.est.7b05825 Environ. Sci. Technol. 2018, 52, 3202−3210

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DOI: 10.1021/acs.est.7b05825 Environ. Sci. Technol. 2018, 52, 3202−3210