Abstract

   As persistent organic pollutants, polychlorinated biphenyls (PCBs)
   accumulate in the bodies of animals and humans, resulting in toxic
   effects on the reproductive, immune, nervous, and endocrine systems.
   The biological and toxicological characteristics of enantiomers of
   chiral PCBs may differ, but these enantioselective effects of PCBs have
   not been fully characterized. In this study, we performed metabolomics
   analysis, using ultra-high performance liquid chromatography tandem
   mass spectrometry (UPLC-MS/MS) to investigate the enantioselective
   toxic effects of PCB95 in zebrafish (Danio rerio) embryos after
   exposure to three dose levels of 0.1, 1, and 10 μg/L for 72 h.
   Multivariate analysis directly reflected the metabolic perturbations
   caused by PCB95. The effects of (-)-PCB95 and (+)-PCB95 were more
   prominent than those of the racemate in zebrafish embryos. A total of
   26 endogenous metabolites were selected as potential marker metabolites
   with variable importance at projection values larger than 1 and
   significant differences (p<0.05). These metabolites included amino
   acids, organic acids, nucleosides, betaine, and choline. The changes in
   these biomarkers were dependent on the enantiomer-specific structures
   of PCB95. Fifteen metabolic pathways were significantly affected, and
   several nervous and immune system-related metabolites were
   significantly validated after exposure. These metabolic changes
   indicated that the toxic effects of PCB95 may be associated with the
   interaction of PCB95 with the nervous and immune systems, thus
   resulting in disruption of energy metabolism and liver function.

Introduction

   Polychlorinated biphenyls (PCBs) are ubiquitous contaminants in the
   environment and can accumulate in the bodies of animals and humans,
   resulting in toxicity to the reproductive, immune, nervous and
   endocrine systems [[36]1]. For example, in Japan 1,684 people were
   poisoned after eating rice bran oil contaminated with PCBs, and more
   than 30 people died in 1968 [[37]2]. Although PCBs became a main
   priority of the Stockholm International Convention in 2001 as a
   persistent organic pollutant (POPs) [[38]3], PCBs present continued
   risks because of their persistence, refractory nature, and ease of
   accumulation.

   Of the 209 PCBs congeners, 78 display axial chirality in their
   nonplanar conformation, and 19 are stable at ambient temperatures.
   Enantiomers of chiral PCBs have different biological and toxicological
   characteristics. Interestingly, the enantiomer fraction (EF) of PCB95
   has opposite effects in fish (EF<0.5) and bivalves (EF >0.5) [[39]4].
   Moreover, enantiomers of PCB139 exhibit different potencies in inducing
   cytochrome P450 activity [[40]5]. Therefore, it is necessary to
   evaluate the enantioselective effects of chiral PCBs on environmental
   problems and their risk to human health and the ecological environment.

   Metabolomics is a relatively new omics technology involving the
   comprehensive measurement of small-molecule metabolites in organisms
   and has been widely used in the study of drug toxicity mechanisms and
   disease. Rat and zebrafish have recently been used as models for
   metabolomic and genomic research [[41]6,[42]7], similarly to research
   on human diseases such as cancer [[43]8] and neurodegenerative diseases
   [[44]9]. Several metabolomic studies of the effects of PCBs in rats
   have demonstrated that PCBs and tetrachlorodibenzo—p-dioxin (TCDD) can
   lead to immune system disturbances, liver and nervous system
   dysfunction, and lipid metabolism perturbation [[45]10]. However, few
   studies have reported metabolomics analysis of PCB enantiomers in
   organisms.

   Therefore, in this study, we used ultra-high performance liquid
   chromatography and tandem mass spectrometry (UPLC-MS/MS) to study the
   enantioselective toxic effects of chiral PCB95 in zebrafish (Danio
   rerio) embryos based on metabolomics.

Materials and Methods

Reagents and materials

   A Milli-Q water purification system (Millipore, France) was used to
   prepare deionized water (18.2 MΩ). HPLC-grade 96% formic acid (MREDA,
   USA) and acetonitrile (Fisher Scientific, USA) were used as solvents.
   Methanol, n-hexane, and dichloromethane were HPLC grade and purchased
   from Fisher Scientific (USA). A mixed standard solution consisting of
   26 compounds, including amino acids, organic acids, amines, and
   choline, was purchased from the National Institute of Metrology
   (China). The PCB95 standard was purchased from AccuStandard
   (purity>98%; USA).

   The enantiomers of chiral PCB95 were separated and quantified by Waters
   2695 HPLC with an ultraviolet detector (USA) at 220 nm and a Lux 5 μm
   cellulose-3 column (250mm×4.6mm×5 μm; Phenomenex, USA) at 30°C. The
   flow rate was 1 mL/min, and the mobile phase was 100% of n-hexane. The
   injection volume was 20 μL. The order of elution of the enantiomers of
   PCB95 was confirmed using an online optical rotation detector
   (IBZMESSTECHNIK, Germany). The first eluted enantiomer was (-)-PCB95,
   and the second enantiomer was (+)-PCB95. The purity of the two
   enantiomers was greater than 98%. This method was validated previously
   in a comprehensive study [[46]11].

Sample collection and preparation

Fish husbandry and embryo collection

   Specific pathogen-free AB line zebrafish (Danio rerio) were cultured in
   a temperature-controlled laboratory (28±0.5°C) with a 14h:10h
   light/dark cycle. The conductivity of the water used for the zebrafish
   was 480+20 μS/cm, and the pH was adjusted to between pH 6.5 and 8.5
   [[47]12]. The fish were fed fresh frozen Artemia (Binhaijun Industry
   Co. Ltd, China) three times daily. Embryos were collected within 0.5 h
   post fertilization (hpf; male/female ratio of 1:1 or 1:2) and were
   rinsed with water.

Exposure of zebrafish embryos

   This study was performed in conformity with Chinese legislation and
   approved by the independent animal ethics committee at the Chinese
   Academy of Agricultural Sciences.

   Chemical exposure was initiated at 5–6 hpf. The embryos were randomly
   allocated to three exposure groups: the racemic group, (-)-PCB95 group,
   and (+)-PCB95 group. The three exposure groups were each treated with
   three dose levels (high, middle, and low), yielding a total of 10
   treatment groups, including one control group. All of the treatment
   groups were performed in ten replications (10 plastic petri dishes per
   group), with 20 embryos per sample and 200 embryos per treatment group.
   Zebrafish embryos were exposed for 72 h at 28 ± 0.5°C with a 14 h: 10 h
   light/dark cycle in constant temperature incubator. The treatment
   volume is 40 mL per petri dish, and the exposure solutions were
   completely renewed every 24 h [[48]13]. Acetone was used as the solvent
   to prepare the stock solution, and the solvent concentration was 0.01%.
   The high-, middle-, and low-dose test solutions were 10, 1 and 0.1 μg/L
   respectively, and the control was only treated with 0.01% acetone.
   After the 72 h treatment, the embryos were collected in 1.5 mL tubes,
   washed three times, and snap-frozen in liquid nitrogen, and then stored
   at -80°C until extraction.

Sample preparation

   The embryo samples were thawed at room temperature and thoroughly
   homogenized in 100 μL of cold methanol on ice. Next, 140 μL of cold
   methanol, 195 μL of distilled water, and 240 μL of dichloromethane were
   added to the homogenate. The samples were then shook for 1 min,
   incubated on ice for 10 min, and centrifuged at 12000× g for 5 min at
   4°C to remove protein and tissue debris [[49]14,[50]15]. After
   centrifugation, 100 μL of the upper polar layer was transferred to a
   1.5-mL glass vial and diluted 10-fold with methanol/water (50:50, v/v)
   containing 0.25% formic acid for UPLC-MS/MS analysis.

   To evaluate the reliability of the preparation method, a standard
   solution containing 26 different types of compounds was added to blank
   extraction solvent at concentrations of 20, 100 or 200 μg/L before
   preparation. A quality control (QC) sample was prepared by mixing equal
   volumes from each embryo sample after preparation. The QC sample and
   the standard solution were analyzed before and during the sequence to
   estimate the method reliability.

UPLC-MS/MS analysis

   UPLC-MS/MS analysis was performed using a Waters UPLC system with a
   QTrap 6500 mass spectrometer (AB SCIEX, USA) in positive ion mode. The
   secondary mass spectrum information for 76 small polar compounds
   selected from the MassBank ([51]www.massbank.jp/) database based on
   references was used for the MRM scanning module. The prepared samples