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Mutagenesis vol. 28 no. 2 pp. 227–232, 2013
Advance Access publication 21 January 2013
doi:10.1093/mutage/ges076
Micronucleus induction in the bone marrow of rats by pharmacological mechanisms.
I: glucocorticoid receptor agonism
Julie E. Hayes, Ann T. Doherty, Michelle Coulson, John
R. Foster, Peter T. Cotton and Michael R. O’Donovan*
AstraZeneca R&D, Mereside, Alderley Park, Macclesfield SK10 4TG, UK
*To whom correspondence should be addressed. Tel: +44 1625 513749; Fax:
+44 1625 231281; Email: [email protected]
Received on September 17, 2012; revised on November 8, 2012; accepted on
November 19, 2012
A novel selective glucocorticoid receptor (GR) agonist,
AZD2906, was found to increase the incidence of micronucleated immature erythrocytes (MIE) in the bone marrow of rats given two oral doses at the maximum tolerated
level. Because GR agonists as a class are considered
not to be genotoxic and AZD2906 showed no activity in
the standard in vitro tests or in vivo in a rat liver comet
assay, investigative studies were performed to compare
AZD2906 with a reference traditional GR agonist, prednisolone. Emphasis was placed on blood and bone marrow
parameters in these studies because GR activation has
been reported to induce erythropoiesis which, in turn, is
known to increase MIE in the bone marrow. Both compounds induced almost identical, small increases in micronucleus frequency at all doses tested. Directly comparable
changes in haematological and bone marrow parameters
were also seen with significant decreases in lymphoid cells
in both compartments and significant increases in numbers of circulating neutrophils. Although no evidence of
increased erythropoiesis was seen as increased immature
erythrocyte numbers either in the blood or in the bone
marrow, histopathological examination showed focal
areas in the bone marrow where the erythroid population was enriched in association with an atrophic myeloid
lineage. This could have been due to direct stimulation of
the erythroid lineage or a secondary effect of myelosuppression inducing a rebound increase in erythropoiesis
into the vacant haematopoietic cell compartment. It was
concluded that the increased MIE frequencies induced
by both AZD2906 and prednisolone are a consequence of
their pharmacological effects on the bone marrow, either
by directly inducing erythropoiesis or by some other
unknown effect on cellular function, and do not indicate
potential genotoxicity. This conclusion is supported by the
lack of carcinogenic risk in man demonstrated by decades
of clinical use of prednisolone and other GR agonists.
Introduction
Corticosteroid drugs such as prednisolone and dexamethasone
have been in clinical use for a considerable time and there has
been no suggestion that, as a class, they present any genotoxic
or carcinogenic risk. Steroids in general are considered not to
be genotoxic and even the genotoxic potential of oestradiol,
oestrone and ciproterone acetate is believed to be irrelevant
under normal physiological and therapeutic conditions (1).
There appear to be very few studies on the genotoxicity of
corticosteroids in the open literature, probably because they
are generally considered to be inactive, and a relatively recent
review (1) found only a single paper which reported that
dexamethasone induced chromosome aberrations in human
lymphocytes in vitro and increased micronuclei in the bone
marrow of mice (2). It was surprising, therefore, when a novel
selective glucocorticoid receptor (GR) agonist AZD2906,
discovered jointly by AstraZeneca and Bayer HealthCare
Pharmaceuticals, which had given negative results in a bacterial
mutation test and mouse lymphoma Tk assay, was found to
increase micronucleus frequency in the bone marrow of rats.
Unpublished observations within the pharmaceutical industry
indicated that this may be a class effect and a subsequent
review of the US product listings for traditional GR agonists
found that the warning labels for a number, including
betamethasone valerate (Luxiq; 2002), clobetasol propionate
(Olux-E; 2006), fluocinonide (Vanos; 2006) and triamcinolone
acetonide (Trivaris; 2008), stated that positive results had been
seen in rodent bone-marrow micronucleus tests. The CDER
Pharmacology review (3) for ciclesonide contains data showing
weak, but clear, increases in micronuclei in the bone marrow
of mice given high oral doses and comparable increases were
also seen with dexamethasone and budesonide included as
reference agents. Ciclesonide was subsequently shown not to
increase tumour incidence in mouse oral and rat inhalation
carcinogenicity studies and from these data it was concluded
that its effect on the mouse bone marrow was a consequence of
GR agonism and did not predict carcinogenic potential.
The purpose of the present study was to examine the effect
of a reference traditional GR agonist, prednisolone, on bonemarrow micronucleus frequency and a range of haematology
and bone marrow parameters in the rat for comparison with
AZD2906 and to make publicly available definitive data to
show them to be a pharmacological class effect.
Materials and methods
All chemicals and reagents were purchased from Sigma (Dorset, UK) unless
specified. The chemical structures of AZD2906 and prednisolone, together
with their IC50s at the GR receptor, are shown in Figure 1.
Animal husbandry
Male Wistar Han rats (substrain HsdHan) were obtained from Harlan UK
and housed three or four per cage. All animals were ~10 weeks old at dosing. Environmental controls were set to maintain conditions of 19–23°C and
40–70% relative humidity, with a 12-h light/dark cycle. All animals were
treated in accordance with approved UK Home Office licence requirements.
Bone marrow micronucleus tests
For both prednisolone and AZD2906, rat bone marrow micronucleus tests
were performed according to current Organisation for Economic Cooperation
and Development (OECD) guidelines, except that concurrent positive control
groups were not included, in the standard protocol in use at AstraZeneca.
Groups of seven male rats were given two doses of the vehicle control or
test compound 24 h apart and killed 24 h after the second dose by halothane
administration and cervical dislocation. A femur was removed from each rat,
the ends clipped to expose the bone marrow and smears prepared and stained
essentially as described by Tinwell and Ashby (4). The femoral cells were
© The Author 2013. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society.
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227
J. E. Hayes et al.
O
O
O
N
H
N
N
OH
OH
HO
H
N
H
H
O
O
F
AZD2906
IC50 values
Human PBMC
Human whole blood
Rat PBMC
Rat whole blood
Prednisolone
= 2.2 nmol/L
= 41.6 nmol/L
= 0.3 nmol/L
= 7.5 nmol/L
Human PBMC
Human whole blood
Rat PBMC
Rat whole blood
= 44.6 nmol/L
= 159.6 nmol/L
= 13.0 nmol/L
= 50.1 nmol/L
Fig. 1. Structures of AZD2906 and prednisolone with their IC50 values at the glucocorticoid receptor determined in whole blood and isolated peripheral blood
mononuclear cells (PBMC) from rat and man. Note: both compounds are highly protein bound in both species.
flushed out using foetal bovine serum containing 25 mmol/l EDTA, filtered
through a 150 μm bolting cloth and centrifuged at 200g for 5 min. The
supernatant was removed, the cell pellet carefully mixed by repeated aspiration
with a Pasteur pipette and a small drop spread onto a clean microscope slide,
air dried and then fixed with methanol. After drying again, slides were dipped
in fresh phosphate buffer pH 6.4, stained with 12.5% acridine orange and then
rinsed in two changes of fresh phosphate buffer and air dried. Prior to analysis,
slides were coded, wet mounted with phosphate buffer and examined blind
by fluorescence microscopy at ×400 magnification. Identification of immature
erythrocytes (IE), mature erythrocytes (E) and micronucleated erythrocytes
(MIE) was described by Hayashi et al. (5).
Initially, for each animal, 2000 IE were scored for the incidence of micronuclei and the ratio of immature to mature erythrocytes (IE:E) was determined in
1000 cells. With AZD2906, the analysis was extended by scoring an additional
4000 IE to give a total of 6000 IE for each animal (6). In order to act as a quality
control procedure, six bone marrow slides from rats treated with cyclophosphamide at 20 mg/kg (72 μmol/kg) in a previous study were included prior to
staining and coding all the slides (7).
AZD2906
AZD2906 (purity 96.5%) was synthesised at AstraZeneca R&D. It was suspended in 0.5% w/v hydroxypropyl methylcellulose and 0.1% Tween 80 in
50 mM citrate buffer of pH 4.0 and groups of seven rats were given two oral
doses of the vehicle, 5, 25 or 50 mg/kg. A previous study had shown 50 mg/kg to
be the maximum tolerated dose. After increases in bone marrow micronucleus
frequency were seen, a subsequent study was performed to investigate possible
haematological and bone marrow changes. Groups of eight male rats were again
given two oral doses of the vehicle, 5 or 25 mg/kg and killed 24 h after the
second dose. Blood samples (0.5 ml into EDTA) were taken for analysis of the
following haematological parameters: erythrocytes, haemoglobin, haematocrit,
mean red cell haemoglobin, mean red cell haemoglobin concentration, mean red
cell volume, red cell distribution width, reticulocytes, platelets, leucocytes, neutrophils, lymphocytes, monocytes, basophils, eosinophils, large unstained cells.
One femur was removed for flow cytometric analysis of the following bonemarrow parameters: erythroid cells per femur, percent erythroid cells, lymphoid cell per femur, percent lymphoid cells, myeloid/erythroid ratio, myeloid
cells per femur, percent myeloid cells, percent red blood cells, total nucleated
cells per femur, percent total nucleated cells.
The second femur was prepared for microscopic pathological assessment as
follows. The femur, attached to the tibia, was removed from each rat, dissected
free of as much skin and muscle as possible and the distal ends of both bones
were cut with bone forceps to expose the inner marrow cavity for fixation.
The entire femorotibial joint was then immersed in approximately 10× its own
volume of 10% phosphate-buffered formalin at pH 7.4. The joint was allowed
to fix for approximately 10 days before being washed and then immersed in a
decalcifying fluid composed of equal parts of 40% formic acid and 10% acetic
acid. The decalcifying fluid was changed every 2 days until, by inspection, the
bone was soft enough to allow slicing with a scalpel without the obvious presence of hard bone. This occurred after approximately 8–10 days of immersion
in decalcifying fluid. The femorotibial joint was then trimmed to fit an embedding cassette and washed in running tap water, dehydrated in graded ethanol
solutions and embedded whole in paraffin wax following clearing in xylene.
The dehydration and embedding schedule was standard for this tissue.
228
Slices of liver and thymus, 3–4 mm thick, were sampled and fixed in the
same fixative as described above for the femorotibial joint, for a minimum of
7 days. The samples were then dehydrated and embedded in paraffin wax using
standard processing schedules.
About 4–5µm thick longitudinal wax sections from femorotibial joints
and transverse sections of liver and thymus were prepared on a microtome,
dewaxed and stained with haematoxylin and eosin stain before being examined
by light microscopy.
Prednisolone
Prednisolone (purity 99%) was suspended in 0.5% hydroxypropyl methylcellulose/0.1% w/v polysorbate 80, groups of seven rats were given two oral doses
of the vehicle, 500, 1000 or 1500 mg/kg and then killed 24 h after the second dose. A previous study had shown 1500 mg/kg to be the maximum tolerated dose. The incidence of MIE in the bone marrow and the haematological
and flow cytometry bone marrow parameters were analysed as for AZD2906.
Because the dose-setting study had shown some evidence of gastrointestinal
ulceration, at necropsy the thoracic and abdominal cavities of all animals were
examined macroscopically.
Statistical analysis
Prior to analysis of the micronucleus tests, the MIE counts were subjected
to an average square-root transformation and transformed counts were analysed in all cases. Analysis of variance, with appropriate linear contrasts,
was used to test for dose-related trends in MIE and was determined in
the following way. If a significant increase was observed across all test
doses, then the trend test was reapplied in a cascade—first excluding the
top dose, then the intermediate dose(s) and finally the control and the lowest dose. This closed testing procedure stops when either a non-significant
effect is detected or all dose levels have been tested. Positive controls were
excluded from this analysis. Positive controls were compared with the concurrent vehicle controls using Fisher’s exact test and was one sided at the
5% level.
Haematology and bone marrow data were analysed pairwise using Shirley’s
test with significance at the 5% level.
Results
Bone-marrow micronucleus frequency
AZD2906 was found to give biologically significant increases
in MIE at all doses after analysis of the standard 2000 IE.
The increases exceeded the concurrent and historical control
values but, because there was inter-animal variability in the
groups given 5 or 25 mg/kg, and also because the results were
unexpected at that time, the analysis was extended to score a
total of 6000 IE. This confirmed that statistically significant
(p <0.001) increases greater than 3-fold the concurrent control
value were seen at all three dose levels (Table I; supplementary
Appendices 1 and 2, available at Mutagenesis Online).
Glucocorticoid receptor, rat, bone marrow, micronucleus
Table I. Summary of bone marrow micronucleus data for rats given two oral
doses of AZD2906 or prednisolone
Table II. Summary of haematology and bone marrow flow cytometry for rats
given two oral doses of AZD2906 or prednisolone
Treatment
Parameter
Vehicle
AZD2906 5 mg/kg
AZD2906 25 mg/kg
AZD2906 50 mg/kg
Cyclophosphamide 20 mg/kga
Vehicle
Prednisolone 500 mg/kg
Prednisolone 1000 mg/kg
Prednisolone 1500 mg/kg
Cyclophosphamide 20 mg/kga
Mean number of MIE
per 2000 IE
per 6000 IE
Mean IE, %
1.6
5.0
5.6
4.4
56.6
2.6
5.9*
6.4**
6.6**
48.7
5.0
16.7***
17.6***
17.1***
178.3***
nd
nd
nd
nd
nd
66.3
65.1
64.6
58.4
65.3
63.7
59.9
57.8
52.6
58.3
Bone marrow sampled 24 h after the second of two doses given 24 h apart.
nd, not determined.
Statistically significant: * p<0.05; ** p<0.05; *** p<0.001.
a
Slides prepared from rats treated in a previous study.
Prednisolone gave statistically significant (P <0.05–0.001)
increases in MIE after analysis of 2000 IEs that were above the
concurrent and historical control values at all dose levels. There
was a dose-related reduction in IE:E ratio.
Haematology and bone marrow parameters
AZD2906: Similar changes in haematological parameters
were seen at doses of 5 and 25 mg/kg. There were reductions
(44–55%) in absolute reticulocyte numbers and total white
cell counts (~75%), mostly due to lower lymphocyte numbers
(~95%) although reductions in monocytes (~75%) and
eosinophils (85–100%) were also noted. There were also minor
increases in neutrophil numbers (65–73%). In the bone marrow,
16% and 6% reductions in nucleated cells were seen at 5 and
25 mg/kg, which were attributed to the lower (~60%) numbers
of lymphocytes.
Prednisolone: Similar haematological changes were seen
at all three dose levels. There were reductions (41–57%) in
reticulocyte numbers and total white cell counts (44–69%).
Although few white cell differential counts were obtained
for technical reasons, it is believed that in a few animals, the
reduction in white cell counts were attributable to reduced
lymphocyte numbers as at the 1000 and 1500 mg/kg doses
(supported by the findings in the bone marrow). There were
also some increases in erythrocyte number (5–8%) at all
doses, haemaglobin and haematocrit, and reductions in platelets (15–24%) in the groups given the two highest doses. In
the bone marrow, lower numbers of total nucleated cells (21–
30%) and lymphoid cells (66–74%) were seen at all doses
(Table II; supplementary Appendices 3 and 4, available at
Mutagenesis Online).
Macroscopic autopsy findings
There was no macroscopic evidence of damage to the
gastrointestinal tract of rats given either AZD2906 or
prednisolone.
Histopathology
Histopathology investigation was only conducted on rats
given AZD2906 and doses of 5 or 25 mg/kg/day induced
histological changes in the liver, thymus and bone marrow of
the femorotibial joint.
% Change of AZD % Change of prednisolone
2906
5 mg/kg 25 mg/kg 500 mg/kg 1000 mg/kg 1500 mg/kg
Haematology
White blood cells −75
Lymphocytes
−96
Neutrophils
+65
Reticulocytes
−44
Bone marrow flow cytometry
Total nucleated
−16
cells
Lymphoid cells
−59
−75
−97
+73
−55
−44
nd
nd
−41
−66
−95
+50
−44
−69
−93
+90
−56
−6
−21
−30
−24
−58
−65
−71
−71
Blood and bone marrow sampled 24 h after the second of two doses given
24 h apart.
% Change for each group is the group mean divided by the control group
mean.
n = 8 per group for AZD2906; n = 7 per group for prednisolone (except 2 per
group for lymphocytes and neutrophils).
nd, not determined.
As expected for a chemical interaction with the GR,
AZD2906 induced an accumulation of glycogen in the liver of
animals given 5 or 25 mg/kg/day with no clear dose–response
relationship for either the incidence of the finding (animals
affected) or the severity of the finding (degree of change).
The thymus of rats given AZD2906 for 2 days showed cortical lymphocytic atrophy of a moderate to marked degree, which
displayed a clear dose–response relationship for both incidence
and severity of the change.
Within the bone marrow of the femorotibial joint, there was
a generalised mild atrophy affecting the marrow in the region
of the growth plate (Figure 2), which targeted the myeloid cell
lines more severely such that there was a clear change in the
ratio of retained cells in favour of the erythroid lineages. In the
shaft areas of the bone, the atrophy was less obvious but the
predominance of erythroid to myeloid cells was more clearly
observed, where the erythroid cells persisted, or might even
have increased, following treatment with AZD2906 (Figure 3).
It was not possible to comment specifically on lymphocyte
numbers in the bone marrow sections following treatment
because of the problem of differentiating lymphocytes from
other cell types present in the sections stained with haematoxylin and eosin. Their positive identification would have required
the use of lymphocyte-specific antibodies that do not work in
this laboratory on bone-marrow sections prepared following
the current decalcification process.
Other genotoxicity studies
The results for the rat liver comet assay, mouse lymphoma
Tk and micronucleus tests and the bacterial mutation assays
are included in supplementary Appendices 5–7 (available at
Mutagenesis Online), respectively.
Discussion
The results from this study clearly show that both AZD2906
and prednisolone have comparable effects on the bone marrow
of rats and the maximum increases in micronucleus frequency
over the concurrent control values, 4.0 per 2000 IE, were
exactly the same for both compounds. No-effect levels (NOEL)
were not established and similar increases were seen at all
229
J. E. Hayes et al.
a)
b)
c)
d)
Fig. 2. Marrow atrophy in the sub-growth plate region of the tibia induced by AZD2906. (a) Control marrow: microscope magnification ×10. (b) Control
marrow: microscope magnification ×40. (c) Marrow from AZD2906-treated rat: microscope magnification ×10. (d) Marrow from AZD2906-treated rat showing
retention/hyperplasia of erythroid cells: microscope magnification ×40.
doses of both AZD2906 and prednisolone, presumably because
the blood levels are so far in excess of their IC50s at the GR.
For AZD2906, two doses of 5 and 25 mg/kg gave Cmax values of
643 and 2520 nmol/l and area under the curve (AUC) of 7270
and 36,400 nmol.h/l, respectively, in comparison with the IC50
of 7.5 nmol/l determined in whole rat blood. Similar plateau
responses have also been reported in the mouse with doses of
ciclesonide from 150 to 2000 mg/kg and dexamethasone from
25 to 1000 mg/kg and the maximum increases, 4.1 and 3.3 per
2000 IE, respectively (3), were also remarkably similar to those
in the rat in the present study. It appears that the propensity
to increase micronucleus frequency in rodents is common to
several GR agonists with differing chemical structures.
In terms of the mechanism of action, it appears unlikely that
DNA reactivity is responsible for the effects in the bone marrow
because there are no reliable reports in the literature of any of the
GR agonists considered here giving positive results in standard
in vitro tests for genotoxicity. Prednisolone has been reported
not to be a bacterial mutagen in a limited study (8) and also in a
test using a standard set of tester strains (9). Although it has been
reported to be weakly active in the mouse lymphoma assay (10),
the increases in mutant frequency do not satisfy current criteria
for a positive result (11). Dexamethasone has also been shown to
230
give negative results in the Ames test but to induce chromosome
damage and sister chromatid exchanges in human lymphocytes
(2). Ciclesonide has given negative results in the Ames test,
an assay for mutation at the hprt locus in Chinese hamster
ovary cells and an assay for chromosome aberrations in human
lymphocytes (3). For AZD2906, no evidence of genotoxicity was
seen in vitro in Good Laboratory Practice-compliant bacterial
mutation and mouse lymphoma Tk assays performed to current
International Conference on Harmonisation (ICH) and OECD
guidelines. The pattern of metabolites of AZD2906 in the rat
has been determined and none (nor AZD2906 itself) has any
structural alert for bacterial mutagenicity. Further, a quantitative
whole-body autoradiography study in rats showed no evidence
of tissue retention of radioactivity, with the exception of the
choroid and retinal pigment, indicating a lack of formation of
reactive metabolites and negative results were also obtained in
a rat liver comet assay (supplementary Appendix 5, available at
Mutagenesis Online) using the same doses as those increasing
bone-marrow micronucleus frequency. For both ciclesonide
and AZD2906, potential aneugenicity can be dismissed from
negative results in in vitro micronucleus tests using Chinese
hamster V79 (3) and mouse lymphoma cells (supplementary
Appendix 6, available at Mutagenesis Online), respectively.
Glucocorticoid receptor, rat, bone marrow, micronucleus
a)
b)
Fig. 3. Erythroid retention/hyperplasia induced by AZD2906 in marrow from shaft region of tibia. (a) Control marrow: microscope magnification ×40.
(b) Marrow from AZD2906-treated rat; microscope magnification ×40.
Because of the lack of evidence of genotoxicity with
AZD2906 and other GR agonists, it seems likely that the
increased incidence of micronuclei in the bone marrow is a consequence of pharmacology-related effects and this is supported
by the similarity in the haematology and bone marrow changes
with both AZD2906 and prednisolone (Table II). There was evidence for retention of the erythroid colonies amongst a general
pattern of myeloid atrophy in AZD2906-treated animals. This
may represent an early hyperplasia of the erythroid series and/
or retention of mature, nucleated erythroid precursors within
the marrow, but it was not possible to arrive at this interpretation unequivocally in the histopathological sections presented.
The effects of glucocorticoids on haematopoietic cells are well
documented in the published literature and have recently been
reviewed (12). Activation of the GR induces apoptosis of lymphocytes, eosinophils and monocytes and protects granulocytes
from apoptosis but also stimulates erythropoiesis (13–15).
Treatment of non-anaemic patients with prednisolone is associated with increased erythropoiesis, and patients with Cushing’s
syndrome (excess circulating glucocorticoids) show elevated
haemoglobin and haematocrit (16). Glucocorticoids are also
used in the treatment of various forms of anaemia (13, 17–19).
GR agonists may stimulate erythropoiesis through a number of
mechanisms including the following:
•• A direct effect on the erythroid lineage mediated via the GR.
Erythroid progenitor cells express the GR and treatment with
glucocorticoids has been shown to induce the proliferation
and expansion of erythroid progenitor cells in vitro while
maintaining their colony-forming capacity and delaying terminal differentiation of erythrocytes (15, 20). In the clinic,
children with acute lymphoblastic leukaemia in remission
show reticulocytosis and increased haemoglobin concentration following pulse prednisolone therapy (13).
•• A secondary response due to marked myelosuppression
mediated via the GR. The bone marrow has been shown to
respond to marked reductions in the haematopoietic cell volume with a rebound increase in erythropoiesis.
Although GR-mediated increase in erythropoiesis is a plausible mechanism for the induction of MIE and histopathological
examination revealed areas where the erythroid population
appeared to be enriched, there were no increased numbers of
immature erythrocytes seen in either the bone marrow or the
peripheral blood. In the bone marrow, it is probable that the
number of cells in the areas of erythroid enrichment were not
sufficient to increase significantly the overall number in the
suspension of cells from the whole femur that were used for
flow cytometric analysis. It is also likely that increased numbers were not seen in peripheral blood because the sampling
time, 48 h after the first dose, was before the majority of the
immature erythrocytes produced after dosing had left the bone
marrow. Finally, it is possible that the increase in MIE is not a
consequence of increased erythropoiesis but another, unknown
effect of GR agonism.
In conclusion, several GR agonists have been shown to increase
the frequency of micronuclei in the bone marrow of rodents. The
data here show directly comparable responses to AZD2906 and
prednisolone in the rat and these are also remarkably similar
to those seen with ciclesonide in the mouse (3). Unpublished
information from within the pharmaceutical industry indicates that
the bone marrow responses to GR agonists have been seen with
other compounds in development and it is likely that the negative
micronucleus tests for some of the older marketed compounds
were not performed to current standards. For example, no evidence
of activity was reported in a test with prednisolone farnesylate (9)
but subcutaneous injection was used and the data are not available
for inspection. Finally, it should be noted that because increases in
MIE were seen at all doses of both AZD2906 and prednisolone,
NOEL were not established for either compound. Further work
would be necessary to establish whether the margin between any
intended therapeutic level of AZD2906 and the NOEL in the bone
marrow is similar to those for prednisolone and other GR agonists.
It has been established previously that increased erythropoiesis
following prior toxicity to erythroblasts or by direct stimulation
of division in these cells can result in increases in bone marrow
micronucleus frequency (21). This study with GR agonists
indicates that anti-inflammatory pharmacology modulating the
erythroid lineage in the bone marrow can have the same effect
but without marked effects on simple indicators of erythropoiesis
such as increased numbers of immature erythrocytes in the bone
marrow or circulating reticulocytes.
231
J. E. Hayes et al.
Supplementary data
Supplementary Appendices 1–7 are available at Mutagenesis
Online.
Acknowledgements
AZD2906 was discovered as part of a collaboration between AstraZeneca
R&D and Bayer HealthCare Pharmaceuticals. The authors would like to thank
the following staff in Safety Assessment, AstraZeneca R&D for their contributions: Deborah Adkins (statistics); Catherine Priestley (comet assay); Mick
Fellows (mouse lymphoma tk and micronucleus tests); Sean Evans, Jenny
Molloy, Katie Wood, Nicola Derbyshire, Debbie Smith (excellent technical
assistance).
Conflict of interest statement: None declared.
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