French1 project protocol

Effects of benzene inhalation exposure in male Diversity Outbred (DO) mice   (2015)

French JE, Gatti DM, Svenson KL, Churchill GA
With: Morgan DL, Kissling GE, Shockley KR, Knudsen GA, Shepard KG, Price HC, King D, Witt KL, Pedersen LC, Munger SC




Project protocol - Contents

Workflow and sampling

Step Description Equipment Data collected
1 Blood drawn for pre-exposure measurements Flow cytometer Counts of peripheral blood erythrocytes and reticulocytes with and without a micronucleus
2 Mice subjected to benzene (0 (air), 1, 10, 100 ppm) for 6 h per day, 5 days per wk for a total of 26-28 exposures Inhalation chamber -
3 Mice euthanized by CO2 asphyxiation on the morning following the last exposure - -
4 Blood and bone marrow collected for analysis Flow cytometer Counts of peripheral blood and bone marrow erythrocytes and reticulocytes with and without a micronucleus

Procedures

Procedure 1: Benzene inhalation exposure

Equipment, software, and supplies

  • Whole body inhalation chambers

Reagents and solutions

  • Benzene
  • CO2

Steps

  1. Mice are randomized to each exposure group after weight stratification such that the mean body weight is similar in each group. Pre-exposure measurements are made following blood collection (see below).
  2. Mice are exposed to one of four benzene concentrations (0 (filtered air), 1, 10, 100 ppm by volume) in whole-body inhalation chambers for 6 h/day, 5 days/wk for 26-28 exposures.
  3. Mice are euthanized following the last exposure.

Additional ARRIVE Guidelines

Allocation of Animals to Test Group: Randomization after weight stratification

Procedure 2: Tissue collection and micronucleus assay

Equipment, software, and supplies

  • Heparin-containing Vacutainer (Becton Dickinson and Co., Franklin Lakes, NJ, USA)
  • EDTA Vacutainer
  • Flow cytometer

Reagents and solutions

  • Heat-inactivated fetal bovine serum
  • MicroFlow BASIC kit (Litron Laboratories, Rochester, NY, USA)

Steps

  1. Peripheral blood is collected pre-exposure via the tail vein and stored in a heparin-containing Vacutainer.
  2. At euthanasia (post-exposure), peripheral blood is collected via cardiac puncture into EDTA Vacutainer tubes.
  3. Bone marrow is collected by aspirating the right femur with heat-inactivated fetal bovine serum.
  4. Samples are processed immediately as described for the MicroFlow BASIC kit.
  5. Cells are counted and micronucleus frequency assessed in reticulocytes and mature erythrocytes using flow cytometry.
  6. Reticulocytes are identified by the presence of an active transferrin receptor (CD71+) on the cell surface; CD71-negative cells are identified as mature erythrocytes.
  7. Approximately 20,000 reticulocytes and 106 mature erythrocytes are evaluated for each mouse with one exception: the percentage of reticulocytes is based on 106 mature erythrocytes.

References

Primary References

French JE, Gatti DM, Morgan DL, Kissling GE, Shockley KR, Knudsen GA, Shepard KG, Price HC, King D, Witt KL, Pedersen LC, Munger SC, Svenson KL, Churchill GA. Diversity Outbred Mice Identify Population-Based Exposure Thresholds and Genetic Factors that Influence Benzene-Induced Genotoxicity. Environ Health Perspect. 2015 Mar;123(3):237-45. doi: 10.1289/ehp.1408202. Epub 2014 Nov 6.   PubMed 25376053     FullText