Distribution of 2,2′,5,5′-Tetrachlorobiphenyl (PCB52) Metabolites in Adolescent Rats after Acute Nose-Only Inhalation Exposure

Inhalation of PCB-contaminated air is increasingly recognized as a route for PCB exposure. Because limited information about the disposition of PCBs following inhalation exposure is available, this study investigated the disposition of 2,2′,5,5′-tetrachlorobiphenyl (PCB52) and its metabolites in rats following acute, nose-only inhalation of PCB52. Male and female Sprague–Dawley rats (50–58 days of age, 210 ± 27 g; n = 6) were exposed for 4 h by inhalation to approximately 14 or 23 μg/kg body weight of PCB52 using a nose-only exposure system. Sham animals (n = 6) were exposed to filtered lab air. Based on gas chromatography-tandem mass spectrometry (GC-MS/MS), PCB52 was present in adipose, brain, intestinal content, lung, liver, and serum. 2,2′,5,5′-Tetrachlorobiphenyl-4-ol (4-OH-PCB52) and one unknown monohydroxylated metabolite were detected in these compartments except for the brain. Liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis identified several metabolites, including sulfated, methoxylated, and dechlorinated PCB52 metabolites. These metabolites were primarily found in the liver (7 metabolites), lung (9 metabolites), and serum (9 metabolites) due to the short exposure time. These results demonstrate for the first time that complex mixtures of sulfated, methoxylated, and dechlorinated PCB52 metabolites are formed in adolescent rats following PCB52 inhalation, laying the groundwork for future animal studies of the adverse effects of inhaled PCB52.


Table of Contents
Chemicals S3 Extraction of PCB52 and its hydroxylated metabolites for targeted gas chromatographytandem mass spectrometry (GC-MS/MS) S3 GC-MS/MS analyses S5 Quality assurance and quality control for the GC-MS/MS analyses S5 Extraction of PCB metabolites from tissues for liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis S6 LC-HRMS analysis S7 Processing of LC-HRMS Orbitrap data and figure visualization S8 Table S1.Abbreviations and unique identifiers of the test compounds and analytical standards S9 Table S2.Summary statistics of body and organ weights S10 Table S3.Summary of organ weights adjusted for body weight S11 Table S4.Recoveries (%) of the Ongoing Precision and Recovery standard spiked into matrices from naïve animals in the GC-MS/MS analysis S12 Table S5.Method detection limits (ng) and limits of detection for each tissue type (ng/g) for the gas chromatographic quantifications of PCB52 and 4-OH-PCB52 S13 Table S6.Surrogate standard recoveries (%) from all tissues in the GC-MS/MS analysis S14 Table S7.PCB or metabolite ng/g wet weight detected levels of each compound across tissue and exposure group S15 Table S8.Precursor ions, product ions, and collision energies for each analyte included in the GC/MS/MS analysis S16 Chemicals.The test compound, PCB52, was synthesized and authenticated using a published guideline. 1Details regarding its authentication are reported elsewhere. 2,3 nalytical standards for gas chromatographic analyses, including 3,3',4,4'-tetrachlorobiphenyl (PCB77) and 2,5,2',5'tetrachlorobiphenyl-4-ol (4-OH-PCB52) were synthesized and authenticated as described previously.and 4'-OH-PCB159 were added to all samples as surrogate recovery standards.PCB204 was used as an internal standard to adjust for volume differences between samples.The recovery standard for the LC-HRMS analysis, 4-sulfooxy-3'-fluoro-4'-chloro-biphenyl ammonium salt (3-F-4'-PCB3 sulfate) was prepared and authenticated as reported elsewhere. 7,8 he potassium salt of perfluorooctanesulfonic acid was provided by Thermo Fisher Scientific (Pittsburg, PA).
Extraction of PCB52 and its hydroxylated metabolites for targeted gas chromatographytandem mass spectrometry (GC-MS/MS).PCB52 and its hydroxylated metabolites were extracted with a liquid-liquid extraction protocol from adipose (0.11 ± 0.01 g), brain (0.7 ± 0.08 g), liver (0.5 ± 0.01 g), and lung tissue (0.5 ± 0.01 g), as described. 9, 10Briefly, tissues were homogenized with 3 mL of isopropanol and 1 mL of diethyl ether.Tissue samples were spiked with surrogate recovery standards (20 ng of PCB77 in 200 µL of isooctane and 20 ng of 4ꞌ-OH-PCB159 in 200 µL of methanol).The samples were capped and inverted for 5 min at 40 rpm and centrifuged at 1,690 g for 5 min to facilitate phase separation.The organic phase was transferred to a second tube containing 5 mL of 0.1 M phosphoric acid in 0.9% NaCl solution.The tissue pellets were resuspended with 1 mL of isopropanol and 2.5 mL hexanes-diethyl ether (9:1, v/v).
The samples were vortexed, inverted, and centrifuged as described above.The organic phases were removed and combined with those from the first extraction step.Next, the aqueous phases were re-extracted with 3 mL hexanes-diethyl ether (9:1, v/v), and the organic phases were combined and concentrated to approximately 0.5 mL under a gentle stream of nitrogen.
Five drops of methanol and 0.5 mL of diazomethane (about 5 mmol) in diethyl ether were added to the organic extracts. 9All samples were stored at 4°C for at least 3 h to allow enough time for derivatization.Excess diazomethane was evaporated under a gentle stream of nitrogen in a fume hood.The extracts were reconstituted in 0. µL sulfatase (type H-2 from Helix pomatia, Sigma-Aldrich, Burlington, MA) for 16 h at 37°C in a shaking water bath.Subsequently, 1 mL of 6 M HCl, 5 mL of 2-propanol, and 5 mL of 1:1 hexanes-MTBE mixture (v/v) was added to the serum, intestinal, and cecum content samples.The samples were inverted for 5 min and centrifuged at 1,690 g for 5 min to facilitate the phase separation.Next, the organic phases of each sample were transferred to new glass tubes, and the aqueous phases were re-extracted with 3 mL of hexanes.Next, 3 mL of aqueous 1% KCl was added to the combined organic extracts, and samples were inverted and centrifuged as described above.The organic phase was transferred to a new tube, and the aqueous KCl phase was reextracted with 3 mL hexanes.The combined organic extracts from the samples were evaporated to near dryness, diluted with 0.5 mL hexanes, and derivatized and cleaned up as described for tissues.Blank samples and an ongoing precision and recovery standard were processed in parallel with all samples.
GC-MS/MS analyses.PCB52 and metabolite determinations were conducted using the multiple reaction monitoring setting (MRM) on an Agilent 7890 A GC system equipped with an Agilent 7000 Triple Quad and Agilent 7693 autosampler.Gas chromatographic separations were performed with an SPB-Octyl capillary column (30 m length, 25 mm inner diameter, 0.25 μm film thicknesses: Supelco, Bellefonte, PA); see Table S8 for the precursor ions, product ions, and collision energies for each analyte.Samples were injected in the solvent vent injection mode with a helium (carrier gas) flow of 0.75 mL/min.Nitrogen was used as the collision gas.The following temperature program was used for the separation of PCB52 and its metabolites: Initial temperature of 45°C, hold for 2 min, 100°C/min to 75°C, hold for 5 mins, 15°C/min to 150°C, hold for 1 min,  S2.The method detection limit (MDL) was calculated from method blanks with the formula: 11,12 , where x̅ is the mean of the replicates from the method blanks, t (n -1, 1 -α = 0.99) is the Student's t-test value for the n -1 degree of freedom with 99% confidence level, and SD represents the standard deviation of the replicates.

Extraction of PCB metabolites from tissues for liquid chromatography-high resolution
mass spectrometry (LC-HRMS) analysis.Aliquots of brain (213-338 mg, n=38), cecum (7-33 mg, n=34), intestinal content (25-56 mg, n=38), liver (247-260 mg, n=38), lung (251-258 mg, n=38) and serum (50-283 mg, n=34) were analyzed by LC-HRMS to identify PCB52 metabolites using a modified protocol based on previous studies. 8Tissues were homogenized in a glass tube with 2 mL of Milli-Q water using a TissueRuptor (Qiagen).The homogenates were then spiked with surrogate standards, 3-F-4'-OH-PCB3 and 3-F-4'-PCB3 sulfate (50 ng of each), in acetonitrile.The homogenate was vortexed for 10 s, and 4 mL of acetonitrile with 1% formic acid was added.After adding 200 mg of sodium chloride and 800 mg of magnesium sulfate, the samples were shaken vigorously, inverted for 5 min, and centrifuged at 1181 g for 5 min to facilitate phase separation.The organic phase on the top was passed through hybrid phospholipid solid-phase extraction (Hybrid SPE) cartridges (3 mL, Millipore Sigma, Burlington, MA), which were loaded with 3 g of a mixture of anhydrous sodium sulfate and anhydrous magnesium sulfate (1:1, w/w) and preconditioned with 3 mL of acetonitrile.The aqueous phase was reextracted with an additional 1 mL of acetonitrile, and the organic phase was again passed through the Hybrid SPE cartridge.The Hybrid SPE cartridges were washed with 3 mL of acetonitrile.The combined eluents were evaporated to dryness using a Savant SpeedVac SPD vacuum concentrator with an RVT5105 refrigerated vapor trap (Thermo Scientific, Waltham, MA) at 35°C.The residual samples were redissolved in 300 μL of acetonitrile and transferred to microcentrifuge tubes.
Potassium perfluorooctanesulfonate (PFOS, 50 ng in acetonitrile) was spiked to the samples as a volume corrector.After the solvent was evaporated to dryness using a SpeedVac concentrator, the extracts were reconstituted with 200 μL of mobile phase (H 2 O-ACN-MeOH = 20-40-40, volume %) and kept in a -20°C freezer for 30 min.The extracts were vortexed for 10 s and centrifuged for 10 min at 4°C and 16,000 g to precipitate the protein.The supernatant was transferred to another microcentrifuge tube and centrifuged using the same parameters.The supernatants were transferred to autosampler vials and kept at -80°C until LC-HRMS analysis.

LC-HRMS analysis. Extracts were analyzed at the High-Resolution Mass Spectrometry
Facility at the University of Iowa on a Q-Exactive Orbitrap mass spectrometry (Thermo Fisher Scientific) with an AXQUITY UPLC-C18 column (particle size: 1.7 µM, 2.1 × 100 mm, Waters, Milford, MA, USA).Mobile phases A and B were water and acetonitrile with a 0.3 mL/min flow rate.The pressure range of the chromatographic system was 4000 to 8000 psi.The UHPLC gradient program was as follows: start at 5% B, hold for 1 min, increase linearly to 95% B, hold for 3 min, return to 5% B, and hold for 4 min before the next injection.The injection volume was 2 µL.The mode used on the Q-Exactive Orbitrap Mass Spectrometry was negative polarity.The current and spray voltage were 18.2 µA and 2472 V.The gas flow rate of the auxiliary and sheath were 2 mL/min and 48 mL/min, respectively.The auxiliary and capillary temperatures were 413°C and 256°C, respectively.The analyses were performed in the full scan mode with a range of 85 to 1000 m/z.The full scan resolution setting was 70,000, the autogain control target setting was 1x10 6 , and the maximum interval time (IT) was 200 ms.

S9
N represents the number of samples analyzed.Recoveries are a culmination of three pools of data.[ng/g] 0.3 0.05 0.1 1 MDL, method detection limit, was calculated based on method blanks. 2 LOD, limit of detection, was calculated based on matrix blanks for each tissue using the formula LOD= mean blank + t 0.01,n-1= 8 *SD blank , where mean blank is the mean of nine blank measures, t 0.01,n-1=

S15
Table S7.PCB or metabolite ng/g wet weight detected levels of each compound across tissue and exposure group (n= 6 per sex and exposure).Data are reported as mean (standard deviation).

Figure S1 .
Calibration curve of (A) PFOS, (B) 3-F-4'-PCB3 sulfate, (C) 4-OH-PCB52 and (D) 4-PCB52 sulfate S17 Figure S2.One trichlorinated OH-PCB metabolite was detected by LC-Orbitrap MS in the intestinal content of PCB52-exposed rats S18 Figure S3.One tetrachlorinated OH-PCB metabolite was detected by LC-Orbitrap MS in the intestinal content of PCB52-exposed rats S19 Figure S4.One tetrachlorinated PCB sulfate was detected by LC-Orbitrap MS in the lung of PCB52-exposed rats S20 Figure S5.One trichlorinated OH-PCB sulfate was detected by LC-Orbitrap MS in the serum of PCB52-exposed rats S21 Figure S6.One tetrachlorinated OH-PCB sulfates was detected by LC-Orbitrap MS in the liver of PCB52-exposed rats S22 Figure S7.One tetrachlorinated MeO-OH-PCB was detected by LC-Orbitrap MS in the serum of PCB52-exposed rats S23 Figure S8.One tetrachlorinated MeO-PCB sulfate was detected by LC-Orbitrap MS in the serum of PCB52-exposed rats S24 Figure S9.The relative levels of PCB52 metabolites from LC-HRMS show distinct differences by compartment but not sex in rats exposed for 4 h to PCB52 S25 References S27

2 .
5°C/min to 280°C, and final hold of 5 min.The unknown OH-PCB was quantified using the relative response factor of 4-OH-PCB52.Quality assurance and quality control for the GC-MS/MS analyses.All analyses were performed following established Standard Operating Procedures.Appropriate blank tissue samples were extracted and analyzed in parallel.Surrogate recovery standards (i.e., PCB77, 4'-OH-PCB159, and 3-F-4'-PCB3 sulfate) were spiked into every sample immediately prior to extraction to correct for analytical losses during sample workup and assess the precision and reproducibility of the extraction across the entire study.Average OH-PCB recovery rates, including the range of recoveries and relative standard deviation for each standard, are provided in Table

Figure S2 .
Figure S2.One trichlorinated OH-PCB metabolite was detected by LC-Orbitrap MS in the

Figure S3 .
Figure S3.One tetrachlorinated OH-PCB metabolite was detected by LC-Orbitrap MS in the

Figure S4 .
Figure S4.One tetrachlorinated PCB sulfate was detected by LC-Orbitrap MS in the lung of

Figure S5 .
Figure S5.One trichlorinated OH-PCB sulfate was detected by LC-Orbitrap MS in the serum of

Figure S6 .
Figure S6.One tetrachlorinated OH-PCB sulfates was detected by LC-Orbitrap MS in the liver of PCB52-exposed rats.(A) Chromatograms extracted based on the theoretical accurate mass of the top three high-abundance isotope ions of tetrachlorinated OH-PCB sulfate ([C 12 H 5 O 5 Cl 4 S] -, m/z 400.86173 for the monoisotopic ion) show a peak at 6.78 min.The accurate masses of several high-abundance isotope ions at (B) 6.78 min match the theoretical accurate mass and isotopic pattern (8:10:5) of a trichlorinated compound.The LC-Orbitrap MS analysis was performed in the negative polarity mode.

Figure S7 .
Figure S7.One tetrachlorinated MeO-OH-PCB was detected by LC-Orbitrap MS in the serum of

Figure S8 .
Figure S8.One tetrachlorinated MeO-PCB sulfate was detected by LC-Orbitrap MS in the serum

Figure S9 .
Figure S9.The relative levels of PCB52 metabolites from LC-HRMS show distinct differences

Table S1 .
Abbreviations and unique identifiers of the test compounds and analytical standards used in this study.

Table S2 .
Summary statistics of body and organ weights (n= 6 per sex and exposure).Data in grams are reported as mean ± standard deviation.

Table S3 .
Summary of organ weights adjusted for body weight (n= 6 per sex and exposure).Data are reported as mean (standard deviation).

Table S4 .
Recoveries (%) of the Ongoing Precision and Recovery standard spiked into matrices from naïve animals in the GC-MS/MS analysis.Data are reported as mean (standard deviation).

Table S5 .
Method detection limits (ng) and limits of detection for each tissue type (ng/g) for the gas chromatographic quantifications of PCB52 and 4-OH-PCB52.

Table S6 .
Surrogate standard recoveries (%) from all tissues in the GC-MS/MS analysis.tetra chlorinated biphenyl, was used as a surrogate standard for PCB52.4ꞌ-OH-PCB159 was used as a surrogate standard for hydroxylated PCB metabolites to determine extraction efficiency.3-F-PCB3 sulfate was a surrogate standard to assess the recovery of PCB sulfates from deconjugation experiments.

Table S8 .
Precursor ions, product ions, and collision energies for each analyte included in the GC/MS/MS analysis.