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Graubert1 project protocol

Drug study: ENU-induced tumorigenesis in 20 inbred strains of mice   (2006)

Graubert TA, Watters JW, McLeod HL
With: Fenske TS

Graubert1 animal documentation

Project protocol — Contents
Workflow and sampling
Equipment
Reagents, supplies, and solutions
Procedure
Data
References

Workflow and sampling

Workflow

Test	Procedure performed	ENU Tx dose	Age	Data collected
1	Baseline hematology	-	9 wk	WBC count, % neutrophils, lymphocytes, platelets, hemoglobin, hematocrit
2	Mice are grouped into control-untreated group (n=191) and ENU treated group (n=172)	2 x 100 mg/kg	9-10 wk	-
3	Mice are examine daily for signs of illness	-	10 wk	gross presence of tumor and/or cancer
4	Mice are examined and then necropsied and tissue samples harvested for histology	-	3-16 mo	WBC count, % neutrophils, lymphocytes, platelets, hemoglobin, hematocrit; gross presence of tumor
5	Veterinary pathologist and clinical hematologist independently examined histology slides	-	3-16 mo	microscopic presence of tumor/cancer

Equipment

• Forcyte veterinary cell counter (Oxford Sciences, Inc., Oxford, CT)
• Spectrophotometer
• Microtome for making 5 µm tissue section before staining
• Dissecting kit (forceps, scissors, scalpels)
• Microscope for examination of histological preparations

Reagents, supplies, solutions

• EDTA-containing vacutainer tubes (Becton Dickinson)
• N-ethyl-N-nitrosourea, 1 g ISOPAC container (Sigma N3385)
• 100% Ethanol

Phosphate/Citrate buffer
• 0.1M NaH₂PO₄
• 0.05M Na₃C₆H5O₇
• Inactivating Solution: 20% w/v Na₂S₂O₃ in 0.1N NaOH

• Disposable plastic cuvettes
• 10 and 30 cc syringes
• 18 gauge needle
• 1 cc tuberculin syringes with 25 gauge, 5/8 inch needles

Stock Solutions
• 1M Sodium Citrate:
• 147 g Na₃C₆H5O₇
• up to 500 mL deionized-distilled water (dH2₂O)

1M Sodium Phosphate, monobasic:
• 60 g NaH₂PO₄
• up to 500 mL dH₂O

Working Solutions
• Citrate/Phosphate Buffer:
• 5.0 mL 1M Sodium Citrate (final concentration 0.05 M)
• 10 mL 1M Sodium Phosphate (final concentration 0.1M)
• 85 mL dH₂O
• pH to 5.0 with Phosphoric Acid
• 0.45 µ filter
Note: Citrate/Phosphate buffer before obtaining pH will be approximately 6.0.

Inactivation Solution
• 50 g Na₂S₂O₃ (Sodium Thiosulfate)
• 1 g NaOH
• up to 250 mL with dH₂O

• 10% formalin tissue fixative
• Paraffin for embedding histology sections
• Hematoxylin and eosin (H&E) stain
• May-Grunwald/Giemsa stain
• Microscope slides for making peripheral blood, bone marrow, and spleen touch preps

Acclimation to test conditions

In general all mice are acclimated to the Washington University School of Medicine specific pathogen-free barrier vivarium facility for at least 1 wk before receiving ENU treatment. Treated mice are observed daily for clinical signs of illness such as hunched posture, pallor, and rapid breathing.

Procedure

I. Baseline hematology measurements
a. Baseline and terminal peripheral blood samples are obtained from the retro-orbital sinus or venous plexus using hematocrit tubes and EDTA-containing vacutainer tubes.
b. The peripheral whole blood (PB) is analyzed for complete blood cell count (CBC) with five-part differential using a Forcyte veterinary cell counter.

II. ENU preparation
a. In preparation for the phosphate/citrate buffer: the pH is adjusted to 5.0 with phosphoric acid and then filter sterilized with a 0.45 µm Filtration system.
b. While safely guarded in a chemical hood, 5 mL of 100% ETOH is injected into the ISOPAC container, then the suspension is gently agitated until the ENU goes into solution.
c. The ISOPAC container is buffered with 95 mL of phosphate/citrate buffer while vented with an 18 gauge needle, then mixed well.
d. The concentration of freshly prepared ENU is determined spectrophotometrically before use.

Safety note: Gloves and a lab coat is worn at all times when working with ENU.

III. Spectrophotometric determination of ENU concentration
a. In a disposable plastic cuvette, 100 µL of the ENU suspension is diluted into 900 µL of phosphate/citrate buffer (1:10 dilution).
b. The optical density (OD) at 395 µm spectral wavelength is determine for the above ENU suspension relative to a blank of phosphate/citrate buffer containing a 1:200 dilution of 100% ethanol.
c. The concentration of the ENU in solution is calculated based on an OD395 of about 1.0 from a standard solution of 1.0 mg/mL. Since the ISOPAC containers of ENU do not contain accurate weight amounts, the OD value is used to determine the exact concentration of the working solution.

Safety note: Whenever possible, keep vials of light and pH sensitive ENU covered with aluminum foil, and use within 3 hours of preparing or thawing.

IV. Injection of mice
a. Between 9-10 wk of age, each mouse is weighed to calculate the proper dose (at 100 mg/kg body weight given twice) before treatment is administered.
b. Mice are divided about equally into two cohorts of untreated-controls and ENU treated mice. All mice are observed for baseline appearance and health condition.
c. The appropriate volume of ENU is loaded into a 1 cc tuberculin syringe while working within a chemical hood.
d. The ENU is injected intraperitoneally (i.p.) into each mouse (if calculated correctly, injection volumes should be between 0.2-0.4 mL per mouse). Because of the alcohol content in the ENU solutions, the mice may appear wobbly or lethargic after injection.
e. The ENU treated mice are kept in a designated area and their bedding changed 24 hrs after injection. The contaminated bedding are then placed into a plastic bag containing paper saturated with inactivating solution.
f. All mice are observed daily for any apparent clinical signs including hunched posture, pallor, and rapid breathing. Moribund mice or mice reaching the age of 16 months post injection are sacrificed and necropsied.

V. Disposal of ENU
a. Spills are cleaned immediately and equipment, syringes and gloves that came in contact with ENU are soaked with the inactivating solution and then discarded.
b. Any remaining or extra ENU is inactivated with 20-30 mL of inactivating solution, allowed to stand in the chemical hood exposed to light overnight and then discarded.

VI. Histopathology survey
a. Following daily examination of control and treated mice, mice showing clinical signs and moribund, are sacrificed and necropsied.
b. After peripheral blood and bone marrow cells are harvested, the mice are examined for the gross presence of tumor, and the location and physical characteristics noted.
c. Following complete necropsy, affected tissues from the skin, mammary gland, lungs, bone (marrow cells), liver, gastrointestinal tract, and genitourinary tract are harvested.
d. Harvested tissue samples are fixed in 10% formalin and later embedded in paraffin for histology.
e. Paraffin embedded samples are sectioned at 5 µm thickness and stained with hematoxylin and eosin (H&E).
f. Microscopic examination of the histology slides are examined blindly by veterinary pathologist and clinical hematologist for the presence of malignancy and cellular characterization.

Data collected by investigator

• Complete blood count (CBC) with differentials: white blood cell counts , % neutrophils and % lymphocytes, platelet count, hematocrit, and hemoglobin.

• Incidence of gastrointestinal, hematopoetic (lymphoid and myeloid), and lung (adenoma and carcinoma) tumors/cancers.