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In vitro and in silico analyses for predicting hepatic cytochrome P450dependent metabolic potencies of polychlorinated biphenyls in the Baikal seal Jean Yoo, Masashi Hirano, Hazuki Mizukawa, Kei Nomiyama, Tetsuro Agusa, Eun-Young Kim, Shinsuke Tanabe, and Hisato Iwata Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b03890 • Publication Date (Web): 18 Nov 2015 Downloaded from http://pubs.acs.org on December 1, 2015

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Environmental Science & Technology

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In vitro and in silico analyses for predicting hepatic cytochrome P450-dependent

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metabolic potencies of polychlorinated biphenyls in the Baikal seal

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Jean Yoo†, Masashi Hirano†, Hazuki Mizukawa†§, Kei Nomiyama†, Tetsuro Agusa†,

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Eun-Young Kim‡, Shinsuke Tanabe†, and Hisato Iwata†*

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†Center for Marine Environmental Studies (CMES), Ehime University,

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Bunkyo-cho 2-5, Matsuyama 790-8577, Japan

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‡

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Department of Life and Nanopharmaceutical Science and Department of Biology,

Kyung Hee University, Hoegi-Dong, Dongdaemun-Gu, Seoul 130-701, Korea

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§

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Graduate School of Veterinary Medicine, Hokkaido University,

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Kita 18, Nishi 9, Kita-ku, Sapporo 060-0818, Japan

Present address: Department of Environmental Veterinary Sciences,

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*

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Center for Marine Environmental Studies (CMES), Ehime University

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Bunkyo-cho 2-5, Matsuyama 790-8577, Japan

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Tel/Fax: +81-89-927-8172

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E-mail address: [email protected]

Corresponding author and address: Prof. Hisato Iwata

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1 ACS Paragon Plus Environment


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Abstract

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The aim of this study was to understand the cytochrome P450 (CYP)-dependent metabolic

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pathway and potency of polychlorinated biphenyls (PCBs) in the Baikal seal (Pusa sibirica). In

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vitro metabolism of 62 PCB congener mixtures was investigated by using liver microsomes of

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this species. A decreased ratio of over 20% was observed for CB3, CB4, CB8, CB15, CB19,

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CB22, CB37, CB54, CB77, and CB105, suggesting the preferential metabolism of low

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chlorinated PCBs by CYPs. The highly activated metabolic pathways in Baikal seals that were

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predicted from decreased PCBs and detected OH-PCBs were CB22 to 4′OH-CB20 and CB77 to

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4′OH-CB79. The total amount of OH-PCBs detected as identified and unidentified congeners

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accounted for only a 3.8±1.7 mol % of loaded PCBs, indicating many unknown PCB metabolic

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pathways. To explore factors involved in CYP-dependent PCB metabolism, the relationships

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among the structural/physicochemical properties of PCBs, the in silico PCB-CYP docking

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parameters, and the in vitro PCB decreased ratios were examined by principal component

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analysis. Statistical analysis showed that the decreased PCB ratio was at least partly accounted

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for by the substituted chlorine number of PCBs and the distance from the Cl-unsubstituted

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carbon of docked PCBs to the heme Fe in CYP2A and 2B.

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Abbreviations: 3-methylcholanthrene (3MC), Baikal seal CYP (bsCYP), Complementary DNAs

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(cDNAs), Cytochrome P450 monooxygenases (CYPs), Data Bank of Japan (DDBJ),

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Dichloromethane (DCM), Generalized Born/volume integral (GB/VI), Hydroxylated PCBs

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(OH-PCBs), L-thyroxin (T4), Methyl t-butyl ether (MTBE), Molecular Operating Environment

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(MOE), Open reading frame (ORF), Polychlorinated biphenyls (PCBs), Principal component

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(PC), Toxic equivalents (TEQs)


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1. Introduction

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Polychlorinated biphenyls (PCBs) are ubiquitously distributed in the environment and

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bioaccumulated in higher trophic animals through the food web because of their persistence and

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hydrophobic nature.1-3 Some PCB congeners elicit a broad spectrum of biochemical and toxic

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effects in humans and laboratory animals, including hepatic enzyme induction, acneform

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eruption, carcinogenicity, immunosuppression, and endocrine disruption.4

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Cytochrome P450 monooxygenases (CYPs) form a superfamily of heme-containing

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isoenzymes that play a central role in altering a wide variety of endogenous and exogenous

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compounds in Phase I reactions.5,6 Members of CYP1, 2, and 3 families are involved in

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drug/xenobiotic metabolism and are not only expressed in the liver but also in extrahepatic

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tissues, such as the lung, kidney, and brain.7 For the Phase I metabolism of PCBs, it has been

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reported that isoenzymes of the CYP1A and 2B subfamilies mostly participate in the insertion

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of an OH group into the biphenyl ring of PCB congeners, depending on their structural

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properties; the metabolism of non-ortho chlorine-substituted PCBs with vicinal H atoms at the

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ortho-meta carbons is catalyzed by CYP1A, whereas the metabolism of PCBs with vicinal H

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atoms at the meta-para carbons is mediated via CYP2B.8,9

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Hydroxylated PCBs (OH-PCBs) are formed by the oxidative metabolism of parental PCBs

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catalyzed by CYPs. The structural similarity between OH-PCBs and L-thyroxin (T4) allows

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some of them to bind competitively to thyroid hormone transport proteins, such as transthyretin,

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a thyroxine-binding globulin, and albumin in the blood.10 Selective binding of some OH-PCBs

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to these transport proteins leads to longer half-lives of metabolites in the peripheral circulation.

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Thus, the toxic effects of OH-PCBs are mainly disturbances in thyroid hormone homeostasis

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and the cerebral nervous system.11,12



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OH-PCBs have been detected in the blood and liver of wildlife, but their levels and congener

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profiles are different among species. It has been recognized that this is at least partly due to

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species differences in the metabolic potency of PCBs by CYPs.13-15 In vitro metabolism assays

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have shown that some PCB congeners were metabolized by CYPs in the liver microsomes of

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rats, hamsters, guinea pigs, humans, whales, and seals.9,15-25 These in vitro metabolism assays

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demonstrate that PCBs are degraded in a congener- and species-specific manner by CYPs in

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reaction solutions containing PCBs and liver microsomes. In some cases, OH-PCBs, which

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appear to be oxidative metabolites derived from added PCBs, have been detected in the reaction

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solution. Although in vitro PCB metabolism has been investigated in a variety of species, there

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are only limited numbers of PCB congeners examined and their metabolites identified.

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Lake Baikal has been polluted with a variety of anthropogenic contaminants, including

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PCBs.26,27 The Baikal seal (Pusa sibirica), a top predator in the lake food chain, has been

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exposed to high levels of coplanar (dioxin-like) and non-coplanar (non-dioxin-like) PCBs for

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the last two decades.28-30 In 1987–1988, an outbreak of morbillivirus infection caused the mass

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mortality of Baikal seals.31 Immunosuppression resulting from chronic exposure to

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environmental contaminants including PCBs has been considered a contributing factor to this

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outbreak.32,33 In addition, we recently confirmed the accumulation of OH-PCBs in the livers of

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Baikal seals and suggested the impact OH-PCBs may have on thyroid function.34 It has also

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been suggested that the hydroxylation of coplanar (non- and mono-ortho chlorine substituted)

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and non-coplanar (di-, tri-, and tetra-ortho chlorine substituted) PCBs is enhanced by the

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induction of hepatic CYPs following exposure to dioxins and related compounds.34,35 Moreover,

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we have sequenced the complementary DNAs (cDNAs) of Baikal seal CYP (bsCYP) 1A1, 1A2,

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and 1B1 open reading frames and built their homology models.36,37 However, the contribution of

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these bsCYPs to the metabolism of PCBs has not yet been directly investigated. Thus, we


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investigated the enzymatic potencies of bsCYPs in PCB metabolism.

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Experimental investigation of xenobiotic metabolism mediated by CYP activities is still

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highly resource-demanding, time-consuming, and technically challenging. Thus, much effort

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has recently been applied to develop computational approaches to predict these metabolic

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outcomes.38 In silico molecular docking analysis is an increasingly popular approach for

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predicting the potency of CYP-dependent substrate metabolism, which is performed by

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simulating the interaction of a substrate with a specific CYP active site.39,40 This approach may

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thus enable the screening of numerous substrate candidates for their metabolism by certain CYP

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isozymes, and eventually lead to the understanding of a comprehensive landscape of CYP-

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mediated metabolic pathways.

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The objective of this study was to evaluate the metabolic potential of PCBs by bsCYP

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proteins. We initially prepared microsomes from the liver of the Baikal seal, and the

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microsomes were then incubated with a mixture of 62 PCB congeners. Following incubation,

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we measured the residue levels of PCBs and OH-PCBs in the incubated microsomal solution by

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chemical analyses. Simultaneously, we sequenced cDNAs of bsCYP2A, 2B, and 2C, and

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constructed in silico homology models using their deduced amino acid sequences. Together with

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previously constructed bsCYP1A1, 1A2, and 1B1 homology models,37 the docking of individual

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PCB congeners to these CYP homology models was simulated by in silico analyses. To examine

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the relationship between the in vitro metabolism of PCBs and in silico docking simulation,

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principal component (PC) analysis was performed, and factors underlying the bsCYP-dependent

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metabolism of PCBs were explored.



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2. Materials and methods

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2.1 Chemicals

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Information on 62 PCB congeners used in this study is given in SI.

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2.2 Sample collection Details on the sample collection are given in SI.

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2.3 Microsomal preparation and CYP spectral analysis Liver microsomal fractions were prepared following the method of Guengerich (1982)41 and details are given in SI.

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2.4 In vitro PCBs metabolism assay

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The reaction mixture (1 mL final volume) for the in vitro PCB metabolism assay was

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prepared from incubation buffer (80 mM NaH2PO4, 6 mM MgCl2, 1mM Na2EDTA, pH 8.0), 1

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µM of the 62 PCB congeners (500 ng/mL for each congener) in DMSO (2% DMSO in the

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reaction mixture) and microsomal suspension containing 200 pmol CYP enzymes. The reaction

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mixture was pre-incubated at 37°C for 10 min. The CYP-dependent metabolic reaction was

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started by adding NADPH regenerating solution (50 µL of solution A and 10 µL of solution B)

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(BD Biosciences, NU, USA), and the reaction mixture was incubated at 37°C for 180 min with

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gentle shaking in a water bath. To investigate CYP-independent metabolism, a reaction mixture


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containing 60 µL of incubation buffer instead of NADPH was also prepared. After incubation,

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the metabolic reaction was stopped by the addition of 1 mL methanol. To confirm the

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reproducibility of the results, this metabolism assay was independently carried out three times.

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2.5 Measurements of PCBs and OH-PCBs The measurement of PCBs and OH-PCBs in in vitro PCB metabolism assays was performed according to the method of Nomiyama et al. (2010).42 Details are given in SI.

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2.6 Sequencing of bsCYP2 cDNAs Sequencing of bsCYP2 cDNAs was carried out according to the method of Hirakawa et al. (2011),35 and details are given in SI.

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2.7 In silico analysis

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All in silico analyses were carried out using the Molecular Operating Environment (MOE)

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program (Chemical Computing Group, Montreal, Canada). Details of these in silico analyses are

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given in SI. In order to construct the 3D structure of heme-containing CYP proteins, a total of

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500 generated structures for each bsCYP were obtained by employing the ‘induced fit’ option

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that allows the heme iron to fit into the template structure. The 3D structures of bsCYPs were

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optimized by PFROSST force field with an energy gradient of 0.05. To generate the final model

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structure, the generalized Born/volume integral (GB/VI) model parameters were applied.

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Structures of PCBs were constructed and energy minimized using Rebuild3D with MMFF94x



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force field in the MOE. A total of 500 confirmations for each PCB congener were generated

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using the default systematic search parameters by LowMode MD method. Molecular docking

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simulations were performed to simulate the binding of 62 PCB congeners to bsCYP proteins

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using ASEDock (Ryoka Systems Inc., Tokyo, Japan).

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2.8 Statistical analyses Statistical analyses were performed as described in SI.


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3. Results and discussion

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3.1 In vitro PCB metabolism by bsCYPs

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The protein concentration of Baikal seal liver microsomes prepared in this study was 10.5

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mg/mL. The total CYP content in the microsome was 0.70 nmol/mg protein. We have

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previously reported that this liver sample contained relatively high levels of total 2,3,7,8-

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tetrachlorodibenzo-p-dioxin toxic equivalents (TEQs; 85 pg/g wet wt.) and bsCYP1A1, 1A2,

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and 1B1 mRNAs.35

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After 62 PCB congeners were incubated with liver microsomes and an NADPH regenerating

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system, the amount of residual PCBs and formed OH-PCBs in the reaction mixture was

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measured by GC-MS analyses. To evaluate CYP-independent metabolism, PCBs and OH-PCBs

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in the reaction mixture without an NADPH regenerating system were also analyzed. CYP-

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dependent metabolism of PCBs was determined by subtracting the PCB amount that was

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decreased without an NADPH regenerating system from the PCB amount that was decreased

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with the system. No residual PCBs were detected in non-treated seal liver microsomes as

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control samples. Based on the results of chemical analyses, the decreased ratio of each PCB

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congener, which is defined as the PCB amount decreased in a CYP-dependent manner to the

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PCB amount loaded into the reaction mixture, was calculated (Figure 1). A decreased ratio of

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over 20% was observed for CB3, CB4, CB8, CB15, CB19, CB22, CB37, CB54, CB77, and

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CB105. Results show that low chlorinated congeners are likely to be more preferably

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metabolized than highly chlorinated congeners. In our previous study34, low chlorinated PCBs

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were detected less in Baikal seal livers (Figure S1). The result in this in vitro metabolism assay

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supported the earlier findings in the seal livers. On the other hand, there are large variations in

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the decreased ratio, even among low chlorinated PCB congeners with the same number of

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chlorine substitutions; for example, trichlorinated congeners such as CB19 (2,2′,6-) and CB37



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(3,4,4′-) showed much greater decreased ratios than CB18 (2,2′,5-) and CB28 (2,4,4′-).

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The large decreased ratio (about 30%) of non-ortho coplanar CB77, a model substrate of rat

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CYP1A, agreed well with those reported in in vitro metabolism assays using the liver

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microsomes of a free-ranging grey seal and harbor seal that had been exposed to high levels of

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PCBs.9,43,44 The high metabolic depletion of CB77 may be due to high levels of bsCYP1A1 in

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microsomes,35 as has been suggested by other in vitro metabolism studies9,43,44. For mono-ortho

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coplanar CB105, the decreased ratio (about 40%) seems to be somewhat different from that

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observed in the grey seal microsome study, in which no significant depletion of this congener

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was observed.9 Meanwhile, hepatic microsomes from a harbor seal seemed to metabolize

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CB105,44 supporting the result obtained in this study. Compared to the result of CB77, the

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smaller decreased ratio (less than 3%) of di-ortho noncoplanar CB52, a model substrate of rat

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CYP2B, appeared to be consistent with the results obtained from grey and harbor seals and

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beluga whale.9,25,44

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In the liver of Baikal seals, CB153 was the most dominant congener, followed by CB138,

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CB187, CB180, CB118, CB105, CB99, CB101, CB149, CB110, CB170, and CB199, as shown

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in Figure S1.34 The decreased ratios of these congeners, except CB105, were less than 10%,

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suggesting the high reliability of this in vitro metabolism assay. The CB105 congener is known

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to be one of the dominant components in Sovol, a technical PCB product, and its production

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reached 100,000 tons in the 1940s-90s in the USSR.45,46 Thus, the high residual of this congener

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in the livers of Baikal seals may be due to high and chronic exposure, despite the modest

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metabolism of this congener by bsCYPs.

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OH-PCBs as well as PCBs were detected in the reaction mixture containing an NADPH

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regenerating system, indicating that bsCYPs catalytically produced OH-PCBs from loaded


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parental PCBs (Figure 1). 3Cl and 4Cl OH-PCBs were major metabolites, followed by 5Cl OH-

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PCBs, supporting the large decreased ratios of low chlorinated PCBs in this in vitro study.

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However, the level of low chlorinated OH-PCBs detected in Baikal seal liver samples was lower

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than that of highly chlorinated OH-PCBs (Figure S1). This could be explained by the limitation

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of our in vitro study; the low chlorinated OH-PCBs may be likely to undergo further metabolic

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processes, such as dihydroxylation and phase II reactions in the liver and/or may be efficiently

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excreted from the liver.15 An alternative explanation is that the low chlorinated PCBs may be

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efficiently degraded in the trophic transfer process of the food web, and Baikal seals may

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therefore be less exposed to them. However, PCB analyses of Lake Baikal fish have shown that

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low chlorinated PCBs are enriched in these animals. 27,30

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While 43 OH-PCB congeners were identified by using their respective authentic standards,

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we could not identify 29 congeners because of the lack of standard compounds (Figure 1).

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Among the identified OH-PCBs, 4OH-CB79 was detected in the highest amount, followed by

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4′OH-CB25/4OH-CB31 and 4′OH-CB20. From 62 loaded PCBs and 43 identified OH-PCBs,

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some metabolic pathways were anticipated: CB18 to 4′OH-CB18, CB22 to 4′OH-CB20, CB28

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to 3′OH-CB28 and 4′OH-CB25/4OHCB31, CB70 to 4OH-CB70 and 4′OH-CB72, and CB77 to

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4OH-CB79. Among them, two pathways, CB22 to 4′OH-CB20 and CB77 to 4′OH-CB79, were

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suggested to be notably activated in the seal microsome (Figure S2), based on the high

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decreased ratios (>20%) of these parent PCBs and the presence of a large amount of their

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metabolites (>0.2 pmol). 4OH-CB79 predominantly formed in this in vitro metabolism assay,

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which is supported by other studies; CB77 was preferably metabolized to 4OH-CB79 rather

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than 5OH-CB77 in the hepatic microsomes of 3-methylcholanthrene (3MC)-treated rats16,47 and

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a free-ranging beluga whale25. As expected from the low decreased ratios (

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