Rusyn6 project protocol

Trichloroethylene metabolism and tissue-specific toxicity in males of 7 inbred strains of mice   (2014)

Rusyn I
With: Yoo HS, Bradford BU, Kosyk O, Shymonyak S, Uehara T, Collins LB, Bodnar WM, Ball LM, Gold A

See also: Rusyn6 animal documentation


Rusyn6 Protocol

Project protocol - Contents

Workflow and sampling

Step
Procedure
Data collected *
1
Mice administered (oral gavage) tricholorethylene (TCE) at 600 mg/kg/d or vehicle only for 5 consecutive days
-
2
Mice given drinking water containing 5-bromo-2'deoxyuridine (BrdU) for 72 h prior to sacrifice
-
3 Mice euthanized and tissues harvested 2 h after last treatment (TCE or vehicle only) Body, liver and kidney weights
4 Tissues processed and analyzed Blood chemistry (BUN); TCE metabolites measured in serum, liver, kidney; BrdU-positive cells determined for liver and kidney; KIM-1 measured in kidney; gene expression for selected genes in liver and kidney

*Supplementary data are available for this project (sub-chronic study). See Rusyn6

Equipment and supplies

  • X-gel tubes (Sarstedt, GERMANY)
  • Microcon YM-10 filter centrifugation (Millipore, Billerica MA)
  • Amicon Ultra Centrifugal Filters 10K (Millipore, Billerica MA)
  • Solid-phase extraction cartridge (StrataTM X-AW, Phenomenex, Torrance CA)
  • SpeedVac Concentrator
  • RNeasy Kit (Qiagen, Valencia CA)
  • Spectrophotometer (ND-1000, Nanodrop Technologies, Wilmington DE)
  • Agilent 2000 Bioanalyser
  • cDNA Archive Kit with random primers (Applied Biosystems, Foster City CA)
  • 96-well plates
  • LightCycler 480 (Roche Applied Science, Indianapolis IN)
  • Microscope

Reagents and solutions

  • Trichloroethylene (TCE)
  • 5% Alkamuls EL-620 in saline (vehicle)
  • 5-bromo-2'-deoxyuridine (BrdU)
  • Neutral-buffered formaliln
  • Liquid nitrogen (N2)
  • Difluoroacetic acid (DFA) (internal standard)
  • Trifluoroacetic acid (TFA) (internal standard)
  • [13C2,15N]DCVG (internal standard)
  • [13C3,15N]DCVC (internal standard)
  • Phosphate-buffered saline (PBS)
  • Tissuelyser (Qiagen, Valencia CA)
  • Sulfuric acid
  • Diethyl ether
  • Acetonitrile
  • Ammonium citrate
  • Methanol
  • Ammonium hydroxide
  • Acetic acid
  • Acetate buffer
  • Beta-glucuronidase (Sigma [G0751], St. Louis MO)
  • Hexane
  • HCl
  • Pepsin solution (Dako, Carpinteria CA)
  • Peroxidase blocking solution (Dako, Carpinteria CA)
  • HRP kit (Dako EnVision System, Carpinteria CA)
  • Monoclonal anti-BrdU antibody (Dako, Carpinteria CA)
  • Dako Liquid DAB Substrate Chromogen System (Dako, Carpinteria CA)
  • Anti-KIM-1 antibody (R&D Systems, Minneapolis MN)
  • Secondary goat IgG HRP-conjugated antibody (R&D Systems)

Procedure: Trichloroethylene (TCE) preparation and treatment of mice

    1. TCE solution is prepared in 5% Alkamuls EL-620 in saline.
    2. TCE is administered by oral gavage (600 mg/kg/d) for 5 consecutive days; control animals receive vehicle only.
    3. Treatment and control mice are given drinking water containing 0.2 g/L of BrdU for 72 h prior to sacrifice.
    4. Mice are euthanized 2 h following the last TCE (or vehicle only) treatment.
    5. Blood is collected from the vena cava and centrifuged to prepare serum using Z-gel tubes according to manufacturer's instructions.
    6. Body and organ weights are recorded.
    7. Liver and kidey sections are fixed in neutral buffered formalin for 24 h then embedded in paraffin; remaining tissue is frozen in liquid nitrogen.
    8. Serum and tissue samples are stored at -80°C until analyzed.

Procedure: Quantification of TCE metabolites

Concentrations of trichloroacetic acid (TCA), dichloroacetic acid (DCA), S-(1,2,-dichlorovinyl)-L-cysteine (DCVC), and S-(1,2,-dichlorovinyl) glutathione (DCVG) are determined using HPLC-ESI-MS/MS as detailed in Kim et al. 2009 with some modifications (see below). Trichloroethanol (TCOH) quantification is performed using the method of Song and Ho, 2003 (gas chromatography-mass spectrometry (GM/MS)). The configuration of instruments is identical to that in the mentioned references, but the extraction methods are optimized for each tissue as described below.

TCA and DCA:

    1. Serum (50 µL) was mixed with 100 µL of the internal standards (DFA and TFA, 50 nmol/mL each); proteins are removed by filter-centrifugation (Microcon YM-10) at 14,000xg for 1 h. Lower limit of quantification is 0.04 nmol/mL for DCA and 5 nmol/mL for TCA. Values below the lower limit of quantification are indicated in the downloadable dataset as "??".
    2. Tissue samples (100 mg) are homogenized in 500 µL of 0.01 M PBS (pH 7.4) with 20 µL of internal standards (DFA and TFA, 20 nmol/mL each) using Tissuelyser for 1 min; homogenates are filter-centrigured (Amicon Ultra) at 14,000xg for 1 h; filtrates are acidified with 100 µL of 3% (v/v) sulfuric acid, 2 mL of diethyl ether added and solutions vortexed vigorously for 1 min; upper layer is transferred to another vial and reduced in volume to less than 300 µL under a continuous stream of N2; the reduced sample is transferred to a glass vial insert containing 5 µL of double-distilled water and dried completely; residue is reconstituted in 20 µL of HPLC moblie phase consisting of 70% acetonitrile, 30% 1 mM ammonium citrate. Lower limit of quantification is 0.1 nmol/g for DCA and 8 nmol/g for TCA. Values belowthe lower limit of quantification are indicated in the downloadable dataset as "??".

DCVG amd DCVC:

    1. Serum (50 µL) was mixed with 100 µL of the internal standards ([13C2,15N]DCVG and [13C3,15N]DCVC, 5 nmol/mL each); proteins are removed by filter-centrifugation (Microcon YM-10) at 14,000xg for 1 h. Lower limit of quantification is 0.5 pmol/mL for DCVG and 1 pmol/mL for DCVC. Values below the lower limit of quantification are indicated in the downloadable dataset as "??".
    2. Tissue samples (100 mg) are homogenized in 500 µL of 0.01 M PBS (pH 7.4) with 20 µL of internal standards ([13C2,15N]DCVG and [13C3,15N]DCVC, 25 nmol/mL each) using Tissuelyser for 1 min; homogenates are filter centrigured (Amicon Ultra) at 14,000xg for 1 h; DCVG and DCVC are extracted using a solid phase extraction cartridge (Strata TM X-AW) (extraction cartriges are conditioned with 300 µL of methanol followed by equilibration with 300 µL of water); samples are loaded, washed with 300 µL of water and eluted with 250 µL of basic methanol (pH 10.8); final eluent is collected in 300 µL glass vial inserts and dried in a SpeedVac concentrator before reconstitution with 20 µL of 4:1 water/methanol containing 0.1% acetic acid. Lower limit of quantification is 2 pmol/g for DCVG and 20 pmol/g for DCVC. Values below thelower limit of quantification are indicated in the downloadable dataset as "??".

TCOH:

    1. Tissue samples (30 mg) are homogenized in 500 µL of acetate buffer (pH 4.6) with 1,000 units of Beta-glucuronidase for 1 min, followed by overnight incubation at 37°C; after centrifugation at 14,000xg for 5 min, the supernatant is transferred to a new tube, then mixed with 20 µL internal standard (DCA, 10 mM in methanol) and 550 µL of water/0.1 M sulfuric acid/methanol (6:5:1); the mixture is heated at 70°C for 20 min; after cooling to room temperature, 2.5 mL hexane is added and vortexed for 10 min and centrigued at 2,500xg for 2 min; the upper layer is concentrated under a stream of N2 to less than 20 µL and used for GC/MS analysis as detailed in Song and Ho, 2003. Lower limit of detection is 5 nmol/g. Values below the lower limit of quantification are indicated in the downloadable dataset as "??".

Procedure: Determination of cell proliferation in liver (hepatocellular) and kidney (proximal tubule)

    1. Deparaffinized and rehydrated sections are immersed in 4N HCl and subsequently pepsin solution for antigen retrieval and then incubated in peroxidase blocking reagent.
    2. An HRP kit (DAKO) is used for the detection of BrdU-incorporated nuclei, using monoclonal anti-BrdU antibody (1:200 dilution).
    3. Data are presented as a fraction (percentage) of BrdU staining-positive nuclei either in the centrilobular region (liver) or the tubular epithelium of the renal cortex (kidney) with no fewer than 1,000 nuclei counted per section.

Procedure: Determination of KIM-1 expression in kidney

    1. Formalin-fixed and paraffin-embedded kidney sections are deparaffinized and rehydrated; antigens are retrieved by 4N HCl and pepsin solution afterward.
    2. After peroxidase blocking, immunohistochemical detection is conducted using Dako Liquid DAB Substrate Chromogen System with primary anti-KIM-1 antibody (2 µg/mL in PBS) and secondary goat IgG HRP-conjugated Antibody (1:100 in PBS).
    3. The proportion of positive-stained proximal tubules in the outer medulla is determined under light microscopy.
    4. Data are presented as a fraction (percentage) of proximal renal tubules staining positive for KIM-1 (no fewer than 200 proximal renal tubules counted per kidney section).

Procedure: Gene expression analysis by real-time PCR (RT-PCR)

    1. Total RNA is isolated from tissue samples using an RNeasy kit according to manufacturer's instructions.
    2. RNA concentration and quality are determined using an ND-1000 spectrophotometer and Agilent 2000 Bioanalyser, respectively.
    3. Total RNA is reversed transcribed using random primers and the high capacity complementary DNA archive kit (Applied Biosystems) according to the manufacturer's protocol.
    4. The following gene expression assays (Applied Biosystems) are used for RT-PCR: Ppara, Mm00440939_m1; Acox1, Mm01246831_m1; Cyp4a10, Mm01188913_g1; and Gusb, Mm00446953_m1.
    5. Reactions are performed in 96-well plates (all samples plated in duplicate) using a LightCycler 480.
    6. The cycle threshold (Ct) for each sample is determined from the linear region of the amplification plot; ΔCt values for all genes relative to the control gene Gusb are determined; ΔΔCt are calculated using treated group relative to strain-matched control group means; fold change data are calculated from the ΔΔCt values.

Data collected by investigator

  • body, liver, kidney weights
  • serum BUN
  • TCE metabolite levels in serum, liver, kidney
  • BrdU-positive cells in liver and kidney (cell proliferation assay)
  • KIM-1 expression in kidney
  • gene expression for selected genes in liver and kidney

Values below the lower limit of quantification are indicated in the downloadable dataset as "??".

*Supplementary data are available for this project (sub-chronic study). See Rusyn6



References

    Kim S, Collins LB, Boysen G, Swenberg JA, Gold A, Ball LM, Bradford BU, Rusyn I. Liquid chromatography electrospray ionization tandem mass spectrometry analysis method for simultaneous detection of trichloroacetic acid, dichloroacetic acid, S-(1,2-dichlorovinyl)glutathione and S-(1,2-dichlorovinyl)-L-cysteine. Toxicology. 2009 Aug 21;262(3):230-8. Epub 2009 Jun 21. PubMed 19549554   FullText

    Song JZ, Ho JW. Simultaneous detection of trichloroethylene alcohol and acetate in rat urine by gas chromatography-mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 2003 Jun 15;789(2):303-9. PubMed 12742121