Appendix D - Dossier (PDF) - Tera

date: 20–JUL–2005
5. Toxicity Substance ID: 71–43–2
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Endpoint: other: AhR knock–out mice and benzene–induced
haematoxicity
Method: ANIMALS AND TREATMENTS
Aryl hydrocarbon receptor knock–out mice (AhR–/–) were mated
with C57BL/6 mice over 6 generations to produce AhR +/+,
AhR+/– and AhR–/– genotypes (confirmed by polymerase chain
reaction screening of DNA). In one series of studies,
animals (age 8 wk, n=5–7) were exposed to 300 ppm benzene
vapour (6 hr/d, 5 d/wk) for 2 wk. In another series, mice
were treated with phenol (PH; 75 mg/kg bwt) and hydroquinone
(HQ; 75 mg/kg bwt) by i.p. injection (saline) twice daily at
6 hr intervals for 3 days (calculated as equivalent to 309
ppm benzene vapour, assuming 100% uptake and metabolic
conversion).
BLOOD AND BONE MARROW PARAMETERS
Haematological parameters (WBC, RBC, haemoglobin,
haematocrit, mean corpuscular volume, mean corpuscular
haemoglobin, mean corpuscular haemoglobin) were quantified
on a sample of peripheral blood collected from the orbital
sinus while bone marrow cellularity was assessed using cell
suspensions prepared from femoral marrow. All determinations
employed automated counting systems.
Granulocyte–macrophage colony forming units (CFU–GM;
lymphocyte progenitor cells) were cultured in semisolid
methyl cellulose culture for 6 days prior microscopic manual
counting.
WESTERN BLOT ANALYSIS FOR P21 AND CYP2E1
Protein extracts from femoral bone marrow cells (Yoon et al.
(2001) Exp Hematol 29, 278) and liver (Valentine et al.
(1996) Toxicol Appl Pharmacol 141, 205) were prepared using
published methods. After denaturation and SDS polyacrylamide
gel electrophoresis, the samples were transferred to a
polyvinylidene fluoride membrane, incubated with rabbit
polyclonal antibody for p21 analysis or goat anti–rat
polyclonal antibody for CYP2E1, then conjugated with
horseradish peroxidase secondary antibody. Band densities
were measured using an image analyser.
REVERSE TRANSCRIPTASE PCR FOR AhR mRNA
Total RNA was extracted from liver or bone marrow cells from
AhR+/+ mice (using a preparatory kit: ISOGEN), and reverse
transcribed prior to PCR amplification with oligonucleotide
primers specific for the mouse Ah receptor (28 cycles for
liver, 30 cycles for bone marrow). The amplified samples
were subject to agarose gel electrophoresis.
STATISTICAL METHODS
Results were analysed using two–tailed Student t–tests.
Result: BLOOD AND BONE MARROW PARAMETERS FOLLOWING BENZENE EXPOSURE
Peripheral blood parameters and bone marrow cellularity were
decreased significantly in AhR+/+ and AhR+/– mice following
2 wk exposure to 300 ppm benzene compared with values from
Appendix D: Benzene SIDS Dossier
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