Appendix D - Dossier (PDF) - Tera

date: 20–JUL–2005
5. Toxicity Substance ID: 71–43–2
______________________________________________________________________________
or in levels of granulocyte–macrophage colony forming units
in bone marrow, or expression of p21. The results indicate
that AhR mediates benzene haematotoxicity, at least in part,
through the induction of CYP2E1.
08–APR–2004 (1301)
Endpoint: other: formation of adducts in mice and rats (i.p.
exposure)
Method: ANIMALS AND TREATMENTS
Male B6C3F1, DBA/2 and C57BL/6 mice (30 g) and male F344/Sim
rats (200 g) were used in these studies. Animals received a
single i.p. injection of 14C benzene in corn oil (0.1 uCi in
200 ul corn oil) and were killed (carbon oxide) 0–48 hr
later. Comment: These strains/species were selected since
they had been used previously in cancer/genotoxicity studies
and appear to differ in susceptibility to benzene toxicity.
SAMPLE COLLECTION AND HPLC ANALYSIS
Liver was sampled and immediately placed on dry ice and
stored frozen. Bone marrow was collected from femur and
humerus and stored (as above).
ISOLATION OF DNA AND PROTEIN FROM TISSUE SAMPLES
DNA was isolated from liver or bone marrow after
solubilisation and RNase treatment, followed by
precipitation with isopropyl alcohol. This procedure gave
approx. 100 ug DNA per 100 mg of starting tissue. Protein
was precipitated with perchloric acid after solubilisation,
and protein content determined using the Bradford
microassay.
ACMS ANALYSIS
The radiocarbon content of the protein and DNA extracts was
determined using accelerator mass spectrometry (AMS) after
converting dried samples to graphite and subsequent analysis
using published procedures (Turtletaub et al. (1993)
Postlabeling Methods for Detection of DNA Adducts, IARC,
Lyon, pp 293–300). Comment: AMS measures the ratio 14C
relative to 13C, which was then normalised to 14C/12C using
a standard carbon source. The ratios were converted to mass
of 14C bnzene based upon the specific activity of the
benzene following correction for background.
STATISTICAL METHODS
Z tests were used to examine for differences between AUCs
for adduct formation.
Result: MACROMOLECULAR ADDUCTS IN RATS AND MICE
LIVER
DNA and protein adducts in liver peaked within 0.5 to 1 hr,
while adduct levels in rat liver continued to increase until
6–12 hr post–dosing. Representative results (pg 14C–benzene
adduct per g protein or DNA) are given below:
Protein adducts
Appendix D: Benzene SIDS Dossier
– 628/957 –