Project protocol — Contents
Workflow and sampling
Reagents, supplies, and solutions
*High-performance liquid chromatography/electrospray ionization tandem mass spectrometry
Steps Procedure Time post-treatment Equipment Data collected 1 Male mice (unfasted) of all cohorts are weighed pre-treatment balance scale body weight 2 Control and treated cohorts are given (via gavage) vehicle (control) or TCE 0 - - 3 Naive, control, and TCE treated mice are weighed and blood sample collected; mice are euthanized for liver and kidney histology 2, 8, 24 hrs balance scale body and liver weight 4 Frozen serum are thawed and analyzed for biological markers of tissue injury - chemical analyzer serum ALT, AST, BUN 5 Frozen serum are thawed and analyzed for TCE metabolites - HPLC-ESI-MS/MS* serum DCA, TCA, DCVC, and DCV
- Small rodent dissection pack (scalpel, forceps, scissors, etc.)
- Centrifuge, tabletop
- Freezer: -80°C
• Blood chemistry analyzer: Automatic Chemical Analyzer, Johnson & Johnson's VT250
• High-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) with a Finnigan Surveyor autosampler and pump coupled to a Finnigan TSQ Quantum triple-quadruple mass spectrometer (Thermo Finnigan, San Jose, CA)
• High-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC-ESIMS/MS) with an Acquity UPLC® (Waters, Milford, MA) system coupled to a TSQ Quantum Ultra triple quadruple mass analyzer (Thermo Finnigan, San Jose, CA)
- Gavage system for mice
- Pressurized CO2 gas for euthanasia
- Trichloroethylene (TCE) (2100 mg/kg) in corn oil (vehicle, 10 mL/kg)
- Corn oil (vehicle)
- Needles and syringes
- Z-gel tubes (Sarstedt, Germany)
Acclimation to test conditions
Mice are acclimated to the new facility for at least 2 wks.
I. Treatment, euthanasia, blood collection, and kidney and liver dissection
a. Unfasted male mice are treated (at 9 am) orally by gavage with TCE (2100 mg/kg) in corn oil (vehicle, 10 mL/kg).
b. Final body weight is obtained before mice are euthanized.
c. Mice are sacrificed at 2, 8 and 24 hrs after treatment. The experimental design is selected based on the previous pharmacokinetic analysis of TCE metabolism in male B6C3F1 mice (Kim et al., 2009).
d. The mice are euthanized by fully trained personnel using CO2 gas asphyxiation.
e. Blood samples are collected via the vena cava for determination of tissue damage using serum biomarkers and TCE metabolites.
f. Blood samples collected without anti-coagulant are allowed to clot for ~30 min before they are centrifuged.
g. Serum is prepared by centrifugation using Z-gel tubes according to the manufacturer instructions, separated, stored frozen at -80°C until ready to be analyzed.
h. The frozen serum is completely thawed before testing for serum alanine transaminase (ALT), aspartate aminotransferase (AST), and blood urea nitrogen (BUN) levels using standard blood chemistry analyzer according to manufacturer's instruction.
II. Determination of TCE metabolites in the serum (see Kim et al., 2009 for greater details)
a. Concentrations of DCA and TCA are measured using HPLC-ESI-MS/MS with autosampler and pump coupled to triple-quadruple mass spectrometer.
b. Determination of DCVC and DCVG is also conducted using HPLC-ESI-MS/MS with an Aquity UPLCÂ® system coupled to a TSQ Quantum Ultra triple quadruple mass analyzer using a heat-assisted electrospray ionization source in positive ion mode.
c. Quantification is based on peak areas relative to the stable isotope-labeled internal standards and the calibration curve is prepared in each batch.
d. Limits of detection are determined by a signal-to-noise ratio of 3:1, such that TCA - 0.4 nmol/mL; DCA - 0.01 nmol/mL; DCVG and DCVC - 0.001 nmol/mL.
Data collected by investigator
• Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and blood urea nitrogen (BUN) in naive, control, and TCE-treated groups at 2 h, 8 h, and 24 h post-treatment.
• TCE metabolites in the serum: trichloroacetic acid (TCA), dichloroacetic acid (DCA), S-(1,2-dichlorovinyl)-L-cysteine (DCVC), and S-(1,2-dichlorovinyl)-L-glutathione (DCVG) in naive, control, and TCE-treated groups at 2 h, 8 h, and 24 h post-treatment.
• Body and liver weights in naive, control, and TCE- treated groups at 2 h, 8 h, and 24 h post-treatment.
dichloroacetic acid (DCA): a major metabolite of TCE.
S-(1,2-dichlorovinyl)-L-cysteine (DCVC): oxidative metabolite of TCE.
S-(1,2-dichlorovinyl) glutathione (DCVG): glutathione-conjugated metabolite of TCE.
trichloroacetic acid (TCA): a major metabolite of TCE.
trichloroethylene (TCE): is widely used as an industrial chlorinated organic solvent. It is found in a variety of sources such as indoor and ambient air, soil and groundwater. Human exposure can lead to hepatotoxicity, neurotoxicity, nephrotoxicity, and auto-immune disorders.
Corton JC. Evaluation of the role of peroxisome proliferator-activated receptor alpha (PPARalpha) in mouse liver tumor induction by trichloroethylene and metabolites. Crit Rev Toxicol. 2008;38(10):857-75.
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.
Kim S, Kim D, Pollack GM, Collins LB, Rusyn I. Pharmacokinetic analysis of trichloroethylene metabolism in male B6C3F1 mice: Formation and disposition of trichloroacetic acid, dichloroacetic acid, S-(1,2-dichlorovinyl)glutathione and S-(1,2-dichlorovinyl)-L-cysteine. Toxicol Appl Pharmacol. 2009 Jul 1;238(1):90-9. Epub 2009 May 3.
Pogribny IP, Rusyn I, Beland FA. Epigenetic aspects of genotoxic and non-genotoxic hepatocarcinogenesis: studies in rodents. Environ Mol Mutagen. 2008 Jan;49(1):9-15.