PNNL-13501 - Pacific Northwest National Laboratory

Approach
Hematopoietic (bone marrow) progenitor cell populations
will be cultured from CBA/Ca mice according to Farris
et al. (1997) and Green et al. (1981). The CBA/Ca mouse
was chosen since it is a useful model for benzene-induced
leukemia and will be of relevance for understanding the
human disease process.
In brief, the CFU-S were cultured in conditioned
semisolid agar media containing macrophage-colony
stimulating factor, interleukin 3, and incubated for up to
12 days at 37°C with various combinations of benzene
metabolites (phenol and hydroquinone). At
approximately 12 hours prior to counting, the cells were
overlaid with a solution of 2-(4-iodophenyl)-3-
(4-nitrophenyl)-5-phenyltetrazolium chloride) that is
metabolized by viable cells forming a red color for
macroscopic quantitation. The CFU-E progenitor cells
were cultured in a methycellulose semisolid medium
containing 30% fetal bovine serum, 1% bovine serum
albumin, and colony stimulating factors erythropoietin as
previously described (Farris et al. 1997). Cultures were
incubated at 37°C for about 2.5 days and examined by
light microscopy, and cell aggregates were scored as
colonies. The CFU-GM assay was conducted using the
agar culture technique described by Farris and Benjamin
(1993). Cultures were incubated for up to 7 days at 37°C
with various combinations of benzene metabolites and on
day 6, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5phenyltetrazolium
chloride was added to the colonies as
previously described for the CFU-S assay and colonies
were scored. The results of these studies were expressed
as number of colonies formed per two femurs and were
compared to the response from nontreated cultures. The
results were expressed as a percent of control.
Similar studies were conducted by exposing stem cells to
the 60 Co source available at our Laboratory. This gamma
radiation source provides an efficient technique for
exposing cells at a known radiation dose and dose rate.
Studies were also conducted with radiation in
combination with benzene metabolites to generate
preliminary data on potential interactions in these specific
cell types important to the development of both radiation
and benzene-induced leukemia.
Results and Accomplishments
Initial efforts primarily focused on optimizing
experimental conditions for isolation and culturing of the
bone marrow stem (CFU-S) and progenitor cells (CFU-E
and CFU-GM) obtained from naïve mice. Once
conditions were optimized, experiments with radiation or
benzene metabolites were conducted. The preliminary
experiments were designed to determine the doseresponse
for bone marrow cell proliferation following
exposure to gamma radiation ( 60 Co) and are presented in
Figure 2. Cells were exposed to a dose range from 0.01 to
10 Gy. For all three cell populations, a clear doseresponse
relationship was established. However, doses
greater than 1 Gy decreased colony formation to 11 to
16% of the control response. Based on these results, the
radiation doses for the mixed exposures were set at 0.01,
0.1, and 1 Gy.
Average +/- SE
80
70
60
50
40
30
20
10
0
Stem Cell Assay Radiation Exposure
CFU-E
CFU-GM
CFU-S
Control 0.01 Gy 0.1 Gy 1 Gy 5 Gy 10 Gy
Figure 2. Colony formation dose-response for CFU-E,
CFU-GM, and CFU-S cells following in vitro exposure to
radiation ranging from 0 to 10 Gy from an external 60 Co
source
The selection of phenol and hydroquinone concentrations
for in vitro evaluation in the stem cell assay were based
on the results seen in bone marrow cells obtained from
Swiss Webster mice (Corti and Snyder 1998). The results
obtained with the CFU-S cells following exposure to
phenol and HQ at concentrations of 0, 10, 20, and 40 µM
and to a mixture of phenol (40 µM) + HQ (10 µM) are
presented in Figure 3. These results in CFU-S cells are
consistent with the response reported by Corti and Snyder
(1998) with CFU-E cells indicating that hydroquinone is
more cytotoxic than phenol and that the combined
response of hydroquinone and phenol, on CUF-S cells is
significantly greater that additive. However, the
combined response was not seen with the CFU-GM or
CFU-E progenitor cells.
The CFU-S cell response to a combined exposure to both
radiation and benzene metabolites is presented in
Figure 4. Again, the radiation exposure produced a
similar dose-response as seen with the preliminary
radiation studies, although the inhibition at 1 Gy was less
that previously observed. In the absence of radiation
treatment, hydroquinone, phenol, and hydroquinone plus
Biosciences and Biotechnology 39