Rusyn7 project protocol

Toxicokinetics and toxicodynamics of tetrachloroethylene in males of 45 Collaborative Cross strains of mice   (2016)

Cichocki JA, Rusyn I
With: Furuya S, Venkatratnam A, McDonald TJ, Knap AH, Wade T, Sweet S, Chui WA, Threadgill DW




Project protocol - Contents

Workflow and sampling

Step
Procedure
 Equipment
Data collected
1
 Mice administered a single dose of tetrachloroethylene (perchloroethylene; PERC) or vehicle Oral gavage equipment
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2

PERC time points: 1, 2, 4, 12, 24 h; Vehicle time point: 24 h

 - -
3 Mice weighed prior to necropsy Balance Body weight
4 Mice anesthetized and euthanasia performed via exsanguination; tissues harvested Dissection kit and balance
 Organ weights
5 Gas Chromatography/Mass Spectrometry (GC/MS) analysis of PERC and trichloroacetate (TCA) in serum and tissues GC/MS equipment PERC and TCA levels in serum and tissues
6 qRT-PCR for Acox1 and Cyp4a10 Roche Light Cycler 480 Expression levels in liver
7 Western blot for CYP2E1 Western blotting equipment Protein levels in liver

Equipment and supplies

  • Amber glass vials
  • Dissecting kit
  • Balance
  • Centrifuge
  • Z-gel tubes, Sarstedt (Numbrecht, Germany)
  • Stainless steel beads for GC/MS
  • Omni Bead Ruptor
  • DB-1 column (60 m x 0.25 mm, 1.0 µm), Agilent 122-1063 (Santa Clara CA)
  • DB-5MS column (30 m, 0.25 mm, 0.5 µm film thickness), Agilent 122-5536 (Santa Clara CA)
  • Teledyne Tekmar Atomx (purge and trap system)
  • Thermo Trace Ultra gas chromatograph
  • Thermo DSQ II single quadrupole mass spectrometer
  • CellCrusher, Cell Crusher (Cork, Ireland)
  • miRNeasy Kit, Qiagen (Valencia CA)
  • NanoDrop C1000, Nanodrop (Wilmington DE)
  • BioAnalyzer, Agilent (Santa Clara CA)
  • cDNA Archival Kit, Thermo Fisher Scientific (Waltham MA)
  • Taqman probe Acox 1 (Mm01246831_m1)
  • Taqman probe Cyp4a10 (Mm01188913_g1)
  • Taqman probe GusB (Mm00446953_m1)
  • Roche Light Cycler 480, Roche (Indianapolis IN)
  • T-PER Tissue Protein Extraction Kit, Thermo Fisher Scientific (Waltham MA)
  • Qibit, Thermo Fisher Scientific (Waltham MA)
  • Pre-cast 10% acrylamide/bisacrylamide gels, Biorad (Hercules CA)
  • PVDF membranes, TurboBlot, Biorad (Hercules CA)
  • C-digit blot scanner, LI-COR (Lincoln NE)

Reagents and solutions

  • Tetrachloroethylene (perchloroethylene, PERC), Sigma Aldrich (St. Louis MO; Catalog #270393-100ML, Lot #SHBD9374V)
  • Alkamuls-EL620, Solvay (Deptford NJ; Lot #SP8J26X07)
  • Phosphate buffered saline
  • Euthasol, Med-Vet International (Mettawa IL)
  • Chloroform:methanol (1:1)
  • 2-bromobutyric acid
  • H2SO4 in methanol
  • Methyl-tert butyl ether
  • Sodium sulfate
  • Sodium bicarbonate (saturated aqueous solution)
  • Helium
  • N2 gas
  • N2 liquid
  • Halt Protease Inhibitor Single-Use Cocktail (100X), Thermo Fisher Scientific (Waltham MA)
  • Odyssey Blocking Buffer, LI-COR (Lincoln NE)
  • Odyssey Stripping Buffer, LI-COR (Lincoln NE)
  • Rabbit anti-CYP2E1 antibody, Abcam (Cambridge MA)
  • Goat anti-rabbit HRP-conjugated secondary antibody
  • Goat anti-beta-actin antibody
  • Amido black

Procedure: Tetrachloroethylene (perchloroethylene, PERC) exposure and tissue collection

  1. Dosing solutions are prepared fresh daily (and used within 1.5 h of preparation) in amber glass vials with foil-lined septa to minimize adsorption of PERC to the container walls and/or septum.
  2. Mice are administered a single dose of PERC (1,000 mg/kg) or vehicle (5% Alkamuls EL-620 in saline) by oral gavage (10 mL/kg).
  3. For PERC-treated mice, the time points for tissue collection are 1, 2, 4, 12, 24 h post gavage (n=1 mouse/strain/time point); vehicle-treated mice are euthanized only at the 24 h time point (n=1 mouse/strain).
  4. Prior to necropsy, mice are weighed and deeply anesthetized by intraperitoneal injection of Euthasol (3 mL/kg) and euthanasia is performed via exsanguination through the vena cava.
  5. Serum is prepared by centrifugation using Z-gel tubes; other tissues are removed, rinsed in phosphate-buffered saline and weighed.

Procedure: Gas Chromatography/Mass Spectrometry (GC/MS) analysis of PERC in tissues

  1. Tissue levels of PERC are assessed via dynamic headspace GC/MS.
  2. Liver or kidney (25 mg) are added to a tube containing 5 stainless steel beads and 1 mL of 5 µM methanolic ethylbenzene (internal standard).
  3. Tissues are homogenized using the Omni Bead Ruptor and quickly added to 40 mL amber glass vials containing 4 mL water, after which the vials are quickly capped with PTFE-lined septa. Samples are run on the same day as homogenization.
  4. The purge and trap system (Teledyne Tekmar Atomx) is set to purge the system with helium for 15 min, followed by adsorption of analytes onto the trap, and finally thermal desorption into a Thermo Trace Ultra gas chromatograph.
  5. Analytes are separated on a DB-1 column and detected via a Thermo DSQ II single quadrupole mass spectrometer operated in full scan mode; under these conditions, PERC and ethylbenzene elute at 17.2 and 18.5 min, respectively.
  6. Quantitation of PERC and ethylbenzene are based on the ions of m/z 129 and 91, respectively.
  7. The ratios of peak areas of PERC to internal standard of tissue samples are used to quantitate PERC concentrations via interpolation of calibration curves generated by spiking known amounts of PERC into tissue homogenates; the assay is linear from 6 to 2000 nmol PERC per gram of tissue.

Procedure: Gas Chromatography/Mass Spectrometry (GC/MS) analysis of TCA in tissues

  1. The analysis of TCA is modified from USEPA Method (EPA 815-B-03-002).
  2. Liver (100 mg) or kidney (50 mg) are added to tubes containing 1 mL of chloroform:methanol (1:1), 20 µL of 2-bromobutyric acid (550 nmol/mL; internal standard) and 5 stainless steel beads. For serum samples, 50 µL is added directly to a vial containing internal standard and esterifying reagent (see below).
  3. Tissues are homogenized using the Omni Bead Ruptor and allowed to sit at room temperature for 5 min before the addition of 200 µL of LCMS-grade water.
  4. Tubes are centrifuged (15,000 g, 10 min, room temperature) and the top (aqueous) layer is removed and added to 8 mL amber glass vials containing 1.5 mL of 10% H2SO4 in methanol (esterifying reagent).
  5. Vials are heated at 50°C for 2 h and then allowed to cool to room temperature. Methyl-tert butyl ether (2 mL) is added to the vials, which are then vortexed and 3 mL sodium sulfate added (150 g/L).
  6. Layers are allowed to separate for 2-3 min, and then the organic layer is removed and added to 3 mL of sodium bicarbonate; after vortexing, layers are again allowed to separate, and then the organic layer is removed and reduced in volume to less than 50 µL under a steady, gentle stream of N2 gas.
  7. The final extract is placed into amber glass vials with 250 µL glass inserts, capped with screw-tops containing Teflon/Silicone-lined septa, and stored at -20°C for less than one week prior to analysis via GC/MS.
  8. Samples (2 µL) are injected via an HP 7673 autosampler into an HP 6890 GC, with the injector maintained at 210°C.
  9. Analytes are separated on a DB-5MS column (flow 1 mL/min).
  10. The initial GC temperature is 40°C, which is held for 10 min; temperature is ramped to 65°C over the next 10 min, then to 85°C over 2 min, and finally to 205°C over 6 min with a final run time of 28 min.
  11. Under these conditions, TCA and methyl bromobutyrate elute at 21.6 and 23.0 min, respectively.
  12. The single quadrupole MS (HP 5973) is maintained at 290°C and operated in splitless mode.
  13. Ions with m/z 59, 83, 85, 117, 119, 121, 132, and 151-154 are scanned at 0.75 cycles/sec; the assay is linear from 4-1000 nmol/g tissue.
  14. The ratios of peak areas of TCA to internal standard of tissue samples are used to quantitate TCA concentrations via interpolation of calibration curves generated by spiking known amounts of TCA into tissue homogenates; the assay is linear from 4-2000 nmol TCA per gram of tissue.

Procedure: Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) for Acox1 and Cyp4a10

  1. Left liver lobe and kidney samples are pulverized in liquid nitrogen using CellCrusher.
  2. Pulverized tissue (15-20 mg) is used for isolation of RNA using miRNeasy kits according to manufacturer's protocol.
  3. RNA concentration is determined by NanoDrop C-1000 and RNA integrity is determined by BioAnalyzer.
  4. cDNA is synthesized using the high-capacity cDNA archival kit from 2 µg of RNA.
  5. qRT-PCR is performed using 2 µL of cDNA (25 ng/µL) in a 20 µL total reaction volume using Taqman probes (Acox1 and Cyp4a10) on a Roche Light Cycler 480 according to manufacturer's recommendations.
  6. Expression of target probe is normalized to GusB expression.

Procedure: Western blotting for CYP2E1

  1. Total protein is isolated from 15-20 mg of pulverized left liver lobe and kidney using the T-PER Tissue Protein Extraction Kit and Halt Protease Inhibitor Single-Use Cocktail according to the manufacturer's protocol.
  2. Protein content is measured using Qubit.
  3. Each gel contains two reference samples to account for inter-gel variability.
  4. Protein (45 µg) is loaded onto pre-cast acrylamide/bisacrylamide gels, electrophoresed, transferred to PVDF membranes, blocked for 2h at room temperature, and incubated with rabbit anti-CYP2E1 antibody overnight at 4°C.
  5. After washing, membranes are incubated with goat anti-rabbit HRP-conjugated secondary antibody for 1 h at room temperature.
  6. After washing, chemiluminescence is used as a detection method of bands using a C-digit blot scanner according to manufacturer's suggestions.
  7. To ensure equal protein loading membranes are stripped and re-blotted with goat anti-beta actin for 1 h at room temperature; the same protocol is used for detection of beta-actin as for CYP2E1; after detection of beta-actin amido black staining of membranes is employed as additional confirmation of equal protein loading.

Data collected by investigator

  • Body weight
  • Change in body weight
  • Liver weight
  • Liver weight to body weight ratio
  • Kidney weight
  • Liver triglycerides
  • Serum triglycerides
  • PERC levels in liver
  • PERC levels in kidney
  • TCA levels in liver
  • TCA levels in serum
  • TCA levels in kidney
  • CYP2E1 protein levels in liver
  • Cyp4a10 gene expression levels in liver
  • Acox1 gene expression levels in liver