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Carcinogenesis |
Department of Pathobiological Sciences [S. M. H., P. S. M., C. J. C.], Environmental Health Sciences Center [S. M. H., M. C. L., C. J. C., C. R. J.], and Department of Pharmacology [M. C. L., C. R. J.], University of Wisconsin, Madison, Wisconsin 53706; Environmental Health Sciences Center, Oregon State University, Corvalis, Oregon 97331 [W. M. B., W. M. D.]; Institute of Toxicology and Environmental Health, Technical University of Munich, Munich D-80636, Germany [J. T. M. B.]; and National Cancer Institute, Bethesda, Maryland 20892 [F. J. G.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Certain PAHs induce multiple tumor types in rodents, including those of hematopoietic tissues, and reduce the cellularity of the bone marrow and spleen in mice. Both in vivo and in vitro studies demonstrate that PAHs suppress humoral immunity by inhibiting B lymphocyte antibody production in response to foreign antigens. The reduction in plaque-forming cells may be the result of PAHs targeting mature B cells or their precursor cells in the bone marrow (7, 8, 9) .
PAHs exert most of their carcinogenic effects after being metabolized to reactive metabolites, which initiate carcinogenesis by covalently binding DNA (10) . The primary route of metabolic activation involves the formation of dihydrodiol epoxides, which can subsequently cause mutations in growth-sensitive oncogenes, such as the H-ras and p53 genes, leading to malignant transformation of the cell (6 , 11) . The synthetic methylated PAH, DMBA, is converted to the most active 3,4-dihydrodiol-1,2-epoxide carcinogenic metabolite via two separate oxidative steps by cytochromes P-450 (12) . The rate and selectivity of these oxidations vary widely among individual cytochromes P-450 in the P-450 superfamily, which also exhibit organ-specific expression (12) . Because the liver is the primary site of DMBA metabolism, a critical issue is whether target organ toxicity results from pharmacokinetic redistribution of reactive metabolites from the liver, or bioactivation of DMBA within the target organ. This question is particularly important in the bone marrow, where DMBA exerts some of its most toxic effects.
The most active forms of PAH-metabolizing cytochrome P-450s are in the CYP1 family. These enzymes exhibit tissue-specific expression that is increased after Ah receptor activation by environmental contaminants such as polychlorinated biphenyls, dioxins, and PAHs. CYP1A1 is highly active for metabolism of most PAHs, but most tissues express negligible CYP1A1 prior to activation of the Ah receptor (13) . CYP1A2 is relatively inactive in the metabolism of most PAHs, except heterocyclic analogues or amino derivatives, and is largely restricted to the liver (14) . A new member of this family, CYP1B1, is active in PAH metabolism but poorly expressed in the liver, kidney, and lung (15 , 16) . Instead, CYP1B1 is constitutively expressed mainly in extrahepatic mesodermal cells, including those in steroidogenic tissues such as the ovary, testis, and adrenal gland, and in steroid-responsive tissues such as the breast, uterus, and prostate. Multipotential stromal fibroblasts in the embryo and bone marrow exclusively express CYP1B1 (17) . This expression pattern suggests a physiological function for CYP1B1 in these tissues, as has been established in the eye, where CYP1B1 mutations result in human congenital glaucoma (18) .
We have recently generated a CYP1B1-null mouse by disrupting a portion
of the open reading frame in exon 3. These mice do not exhibit any
obvious reproductive or developmental abnormalities (19)
.
However, multiple dosing with DMBA resulted in lymphoblastic lymphomas
in 
70% of wild-type mice but only 7% of CYP1B1-null mice
(19)
. The levels of CYP1A1, which is more active in the
metabolism of DMBA, were far greater than CYP1B1 in the major organs
after DMBA treatment and were similar in wild-type and CYP1B1-null
mice. This suggests that although CYP1A1-mediated metabolism may
dictate the systemic pharmacokinetics of DMBA, activation by CYP1B1 in
target tissues such as the bone marrow may be critical for the toxicity
and carcinogenicity of DMBA.
In a recent study, we demonstrated that multipotential bone marrow stromal cells express high basal levels of CYP1B1 that metabolize DMBA to the mutagenic precursor DMBA-3,4-dihydrodiol (17) . In this study, we have tested the hypothesis that reactive DMBA metabolites generated by CYP1B1 are responsible for a rapid depletion of bone marrow cells and the appearance of preleukemic cells in the bone marrow. We suggest that this mechanism initiates the high proportion of lymphoblastomas that result from repetitive administration of DMBA.
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MATERIALS AND METHODS |
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Animals and Treatments.
C57Bl/6 mice were purchased from The Jackson Laboratory (Bar Harbor,
ME). CYP1B1-null mice were produced and characterized as described
previously (19)
. Mating C57Bl/6 mice with CYP1B1-null mice
generated CYP1B1 heterozygous mice. Animals were housed at the
Association for Assessment and Accreditation of Laboratory Animal Care
International-certified University of Wisconsin-Madison School
of Veterinary Medicine Animal Care Facility and used in accordance with
the NIH Guide for the Care and Use of Laboratory Animals.
C57Bl/6 mice (5–8 weeks of age) were randomly selected and received injections i.p. of 10–200 mg/kg DMBA in olive oil. Control animals received injections of an equivalent volume of olive oil. In subsequent experiments, 50 mg/kg DMBA were used because this dose consistently resulted in peak bone marrow toxicity in wild-type mice but had no effect in CYP1B1-null mice.
Harvesting Bone Marrow Cells.
Mice were sacrificed 48 h after injection with DMBA or oil
vehicle. The femurs, tibias, and humeri were dissected free of muscle
tissue, and the ends were removed with surgical scissors. For total
bone marrow cell counts, cells from both femurs and a single tibia were
flushed from the bones with culture medium using a syringe equipped
with a 25-gauge needle. After centrifugation, RBCs were lysed in ACK
buffer (150 mM NH4Cl, 1.0
mM KHCO3, and 100 mM
Na2EDTA, pH 7.3). Viable cells were identified
and enumerated in a hemocytometer by their exclusion of 0.04% trypan
blue (Fig. 1)
. An aliquot of 105 cells was removed and stained
with 1 µl of FITC-conjugated RB6–8C5, anti-B220 (PharMingen, Inc.),
or sca-1 (PharMingen, Inc.). The standard optics of the Coulter
Profile II were used to separate and measure the fluorescence emissions
from each cell. The data from 2 x 104 cells were collected, transformed to standard
FCS format using Pro2FCS software (Verity Software House, ME),
and quantified using Winmidi 2.7 software (Joe Trotter; Scripps
Institute, La Jolla, CA). The remaining cells were used for DMBA-DNA
adduct analysis. Bone marrow smears were prepared by cutting a tibia
longitudinally, streaking the exposed bone marrow onto a glass slide
with a fine sable hair brush, and allowing the cells to air dry at room
temperature.
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Cell Cultures.
The 70Z/3 preB cell line was purchased from the American Type Culture
Collection (Rockville, MD) and routinely grown in culture medium as
described above. The BMS2 bone marrow stromal cell line
(20)
was generously provided by Dr. Paul Kincade (Oklahoma
City, OK) and maintained in the same tissue culture medium described
above. Primary bone marrow stromal cells were isolated and cultured in
six-well plates as described elsewhere (21)
. Primary
cultures from animals with different genetic backgrounds had similar
cellular compositions, as determined by light microscopic examination.
After 3–5 weeks of culture, the nonadherent cells were removed, and
the remaining adherent stromal cells were used in experiments.
PreB Cell Apoptosis Assays.
Exponentially multiplying 70Z/3 preB cells were centrifuged, counted
with a hemocytometer, and diluted to 106 cells in
a total volume of 2.5 ml culture medium. Primary bone marrow stromal
cell cultures were established in six-well tissue culture plates as
described above and then incubated with 2.5 ml of the 70Z/3 preB cell
suspension. After 24 h of incubation with either 10
µM DMBA or 0.1% vehicle control, the culture medium
containing the suspended preB cells was removed and placed on ice. PreB
cells that remained adherent to the bone marrow stromal cells were
dislodged by gently tapping the plate. The detached preB cells were
collected in ice-cold PBS and combined with the original preB cell
suspensions from the same well. Light microscopy revealed that the bone
marrow stromal cells remained adherent to the well, whereas most of the
preB cells where removed (>99%). The TUNEL assay was used to label
apoptotic preB cells, according to the manufacturer’s directions
(Apoptosis-Fluorescence kit; Promega Corp., Madison, WI). The standard
optics of the Coulter Profile II (Hialeah, FL) flow cytometer was used
to detect and enumerate fluorescent apoptotic cells. The data from
2 x 104 cells were collected,
transformed to standard FCS format using Pro2FCS software (Verity
Software House), and quantified using Winmidi 2.7 software (Joe
Trotter; Scripps Institute).
RT-PCR Amplification of CYP1B1 mRNA.
Bone marrow cells were aspirated from the humeri using a syringe with a
25-gauge needle. Their total RNA was isolated with TRIzol reagent (Life
Technologies, Inc., Grand Island, NY), according to the manufacturer’s
directions. RNA isolated from three mice in the each group was pooled
to increase the working amount of RNA. Total RNA was isolated from 70%
confluent BMS2 cells as a reference for CYP1B1 mRNA expression. The
Access RT-PCR System (Promega) was used to amplify CYP1B1 mRNA using an
upstream (5'-GGCGTTCGGTCACTACTCTG-3') and a downstream
(5'-AGGTTGGGCTGGTCACTCAT-3') CYP1B1 primer. After 40 amplification
cycles (1 min at 94°C, 1 min at 57°C, and 1 min at 72°C), 20 µl
of each PCR reaction mixture were electrophoresed in a 2% agarose gel,
the gel was stained with ethidium bromide, and the PCR products were
visualized with UV light.
Western Immunoblots.
A piece of liver from each mouse was flash-frozen in liquid nitrogen.
Microsomes were prepared from thawed livers as described previously
(22)
. Protein concentrations were determined by the BCA
method (Pierce Chemical, Rockford, IL). Proteins were resolved by
SDS-PAGE electrophoresis, transferred to nitrocellulose, and
immunoblotted for CYP1A1 or CYP1B1 as described previously
(15)
. Immunoblot signals were quantified using a Molecular
Dynamics (Sunnyvale, CA) Personal Densitometer SI and Image QuaNT
software.
DMBA-DNA Adduct Analysis.
Bone marrow cells isolated as described above were lysed, and their DNA
was isolated, digested with nuclease P1, and postlabeled with
[
-33P]ATP, and the mononucleotide adducts
were analyzed by HPLC as described previously (23)
. The
elution time for a standard of DMBA-3,4-dihydrodiol-1,2-epoxide-DNA
adduct was used to identify the corresponding peak in the elution
profiles of bone marrow cell DNA.
Statistical Analysis.
ANOVA analysis was used for statistical comparison of control and
DMBA-treated total bone marrow cell count data for each of the three
mouse genotypes. A minimum of four animals was used for each data
point. PreB cell apoptosis data were analyzed for significance within a
mixed model ANOVA using the SAS statistical software program.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Flow cytometry of bone
marrow revealed a significant decrease (P < 0.05) in RB6–8C5-positive myeloid cells (mean ± SE of
14.6 ± 1.4 x 106
versus 24.4 ± 1.5 x 106) and B220-positive B lymphocytes
(7.9 ± 0.5 x 106
versus 9.3 ± 0.6 x 106) in DMBA-treated versus control
wild-type mice (mean ± SE of three and four mice,
respectively). This level of reduction represents the maximum that can
be attained with DMBA, as evidenced by no further suppression after
treatment with 4-fold higher concentrations of DMBA or at examination
at earlier time points. An increase (P < 0.05) in sca-1-positive stem cells was also noted in DMBA treated
versus control wild-type mice (1.6 ± 0.1 x 106 versus
1.1 ± 0.1 x 106,
respectively). In contrast, no reduction of total bone marrow cells was
observed in CYP1B1-null mice treated with DMBA (Fig. 1)
. CYP1B1
heterozygous mice (CYP1B1+/-) demonstrated an intermediate response
after DMBA treatment. The apparent differences in baseline cell numbers
among the three groups of untreated mice do not necessarily reflect
genotype differences, because the three groups of mice were not
littermates and were not matched for age and weight. Other experiments
with CYP1B1 (+/+) mice demonstrated that comparable variation can occur
among mice from different litters or of different ages. As a result,
the relevant comparisons in Fig. 1
are within, rather than across, the
three genotypes of mice.
Histopathological examination of vehicle-treated CYP1B1-null mouse bone
marrow revealed that they were similar to those of control wild-type
mice (Fig. 2)
. The bone marrow of DMBA-treated CYP1B1-null mice could not be
distinguished from vehicle controls and received a histological grading
of normal (Fig. 2
; Table 1
). The decreased numbers of total bone marrow cells after DMBA
administration in wild-type mice was reflected histopathologically as
markedly hypocellular bone marrow, accompanied by dilated sinusoids
containing mature RBCs. The most severe cell reductions occurred in
those cells belonging to the granulocytic and erythroid series. This
loss of bone marrow cellularity was consistent in all DMBA-treated
wild-type animals and resulted in an average histological grade of 3+
(4+ was the most severe). Along with the reduction in cell density, the
bone marrow of DMBA-treated wild-type mice contained a greater
proportion of large, dark blue-staining cells compared with
vehicle-treated mice. These cellular characteristics are commonly
associated with proliferating blast cells, and all DMBA-treated
wild-type mice received a histological grading of 4+ for blast cells.
The increase in blast cells is consistent with the increased number of
sca-1-positive stem cells noted above. DMBA treatment failed to reduce
the density of bone marrow cells in CYP1B1-null mice or to increase the
number of blast cells. Treatment of CYP1B1+/- with DMBA resulted in an
intermediate response, which is reflected by their histological grading
(Table 1)
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, f and
e, respectively). Cytochemical staining suggests that the
majority of these blast cells were of granulocytic
lineage.5
Although some of these cells were normal proliferating blasts, many
exhibited evidence of dysplasia, as characterized by asynchronous
maturation of the nucleus relative to the cytoplasm. More specifically,
myeloblasts and promyelocytes, which are typically characterized by
their azurophilic granules and round nuclei, were larger and darker
stained than expected. The postmitotic granulocytes also appeared
dysplastic, as distinguished by their enlarged size compared with the
stage of nuclear maturation. In addition, these cells had very
pleomorphic nuclei, as distinguished by their elongate, convoluted, or
lobulated shapes. These cellular morphologies are characteristic
of myelodysplastic bone marrow cells, which are one of the hallmarks of
preleukemia (25)
. Because these myelodysplastic cells
belong to the granulocytic series, the granulocytic:erythrocytic cell
ratios in DMBA-treated wild-type mice were increased (92:8) compared
with vehicle-treated mice (61:39; Table 1
). The bone marrow smears from
DMBA-treated CYP1B1-null mice could not be distinguished from control
smears and, therefore, had a similar granulocytic:erythrocytic ratio as
controls. Collectively, these findings demonstrate that CYP1B1 is
required for the cytotoxic and preleukemic effects of DMBA in the bone
marrow.
Expression of Hepatic CYP1A1 and Bone Marrow CYP1B1.
CYP1A1 metabolizes DMBA more rapidly than CYP1B1 and produces much less
of the 3,4-dihydrodiol metabolite (12)
. We, therefore,
considered the possibility that differences in hepatic CYP1A1 might
influence DMBA-mediated bone marrow toxicity. Liver microsomes were
isolated from control or DMBA-treated mice and immunoblotted for
CYP1A1. Fig. 3A
demonstrates that low levels of constitutive hepatic CYP1A1
are substantially increased after DMBA treatment of wild-type,
CYP1B1+/-, and CYP1B1-null mice. These levels of hepatic CYP1A1 were
similar in all three groups of mice. Hepatic CYP1B1 was not detectable
by immunoblotting in either wild-type or CYP1B1 heterozygous mice.
Elsewhere, we have demonstrated that hepatic CYP1B1 is typically
20–100 times lower than hepatic CYP1A1, even after maximal inducing
conditions (15)
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. This level of CYP1B1 expression was much lower than in
the BMS2 bone marrow stromal cell line. In a previous report, we show
that cultured primary bone marrow stromal cells express 2–3-fold less
CYP1B1 than BMS2 cells, and that none of these cells express detectable
CYP1A1. These differences in CYP1B1 expression are likely attributable
to the enrichment of stromal cells in primary bone marrow cultures
relative to bone marrow in vivo (17)
.
CYP1B1-Null Mice Have Significantly Reduced Levels of Bone Marrow
DMBA-DNA Adducts.
CYP1B1-null mice are resistant to DMBA-induced bone marrow cytotoxicity
and the generation of preleukemic cells. The constitutive bone marrow
expression of CYP1B1 in wild-type mice and the similar hepatic CYP1A1
expression in all strains of mice examined suggest that CYP1B1-null
mice are resistant because the responses require local activation of
DMBA by CYP1B1. In contrast, hepatic metabolism presumably produces PAH
dihydrodiol epoxide metabolites that are stabilized by lipoproteins in
the blood (26)
. Hepatic metabolism should, however, be
independent of the CYP1B1 genotype, because this form is absent in
these livers. To investigate these possibilities, DNA was isolated from
the bone marrow of DMBA-treated and control mice and analyzed for the
presence of DNA adducts. These adducts can provide a measure of the
amount and type of DMBA metabolites present in bone marrow. Dihydrodiol
epoxide DMBA-DNA adducts were detected in the bone marrow of wild-type
mice after DMBA administration, with the 3,4-dihydrodiol-1,2-epoxide
DMBA-DNA adducts accounting for most of the adducts present (Fig. 4)
. This pattern of dihydrodiol epoxide adducts is typical of DMBA
activation by CYP1B1, rather than CYP1A1, which additionally produces
more polar adducts (27)
. Bone marrow from DMBA-treated
CYP1B1 null mice had <5% of the dihydrodiol epoxide DMBA-DNA adducts
present in the bone marrow of DMBA-treated wild-type mice. These
results indicate that CYP1B1-dependent DMBA metabolism is responsible
for >95% of the total bone marrow DMBA-DNA adducts in wild-type mice.
The correlations between these adduct levels, and both the cytotoxic
and preleukemic actions of DMBA, suggest that these effects are
dependent on DMBA-dihydrodiol epoxide production. The presence of
CYP1B1 in bone marrow suggests that this activity, rather than hepatic
metabolism by CYP1A1, is responsible for DMBA activation.
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2% of preB cells were apoptotic when
cocultured with primary bone marrow stromal cells (Fig. 5)
, which was the same amount as preB cells cultured with DMBA in the
absence of bone marrow stromal cells (data not shown). These findings
demonstrate that bone marrow stromal cell expression of CYP1B1 is
required for DMBA-induced preB cell apoptosis.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previous work demonstrated that CYP1B1 in primary bone marrow stromal cells metabolizes DMBA, thus supporting the possibility that the bone marrow toxicity of DMBA may be attributable to local activation (17) . In contrast, dihydrodiol epoxides of benzo(a)pyrene are stabilized by serum lipoproteins, suggesting that hepatic metabolism may be the prime source of toxicity in extrahepatic tissues (26) . The present study indicates that this later mechanism is not effective in causing bone marrow toxicity.
Selective protection from the acute bone marrow toxicity of DMBA parallels our recent finding that CYP1B1-null mice are resistant to DMBA-induced lymphoblastic lymphomas (19) . This resistance of CYP1B1-null mice implicates CYP1B1 as being essential for initiating the carcinogenic effect of DMBA in immune organs. A linkage to leukemia initiation is supported by the present finding of myelodysplastic bone marrow cells in DMBA-treated wild-type mice but not CYP1B1-null mice. Myelodysplastic cells are one of the distinguishing features of preleukemia, a syndrome that greatly increases the likelihood of leukemia development (30) . Others have observed the preleukemic effect of DMBA (25) , and chronic DMBA treatment results in leukemias and lymphomas (31 , 32) .
The carcinogenicity of DMBA has been linked to the covalent binding of DMBA dihydrodiol epoxides to DNA (10 , 33) . There is also evidence that DMBA initiates carcinogenesis without an intermediate dihydrodiol (34) , possibly via DMBA radical cations (35) . The data presented here suggest that the DMBA dihydrodiol epoxide DNA adducts in wild-type mouse bone marrow cause mutations that initiate leukemias and lymphomas. The near absence of bone marrow DMBA-DNA adducts in CYP1B1-null mice, combined with their resistance to DMBA-induced lymphoblastic lymphomas, strongly implicates DMBA metabolism by bone marrow CYP1B1 as being involved in immune cell carcinogenesis in wild-type mice. DMBA metabolites generated by CYP1B1 activity may have also removed immune cells that may be important for eliminating mutant cells generated by carcinogens.
We have demonstrated that bone marrow cells constitutively express CYP1B1 both in vitro and in vivo, and that wild-type and CYP1B1-null mice had comparable amounts of hepatic CYP1A1 after DMBA treatment. Despite these similar levels of hepatic CYP1A1, CYP1B1-null mice are resistant to DMBA-induced bone marrow toxicity, indicating that these sizable levels of CYP1A1 contribute little to this toxicity. Certainly, the conversion of DMBA to the 3,4-dihydrodiol precursor of the most mutagenic metabolite is favored by CYP1B1 relative to CYP1A1 (12) . However, this selectivity does not apply to benzo(a)pyrene, which is readily activated to mutagenic precursors by CYP1A1 (13) , but exhibits much less bone marrow toxicity than DMBA.6 That bone marrow CYP1B1 is sufficient for inducing toxicity was further established by in vitro studies that show DMBA-induced preB cell apoptosis is dependent on bone marrow stromal cell CYP1B1.
The constitutive and Ah receptor inducible levels of CYP1B1 and CYP1A1 may also be important for target organ toxicity. For instance, CYP1B1 is constitutively expressed in organs that are targets for the carcinogenicity of DMBA (uterus, ovary, skin, and bone marrow), whereas only CYP1A1 is expressed in liver, and this is dependent on Ah receptor activation (15, 16, 17) . Because DMBA only modestly activates the Ah receptor, low levels of hepatic CYP1A1 are induced relative to potent Ah receptor ligands, such as benzo(a)pyrene. Thus, appreciable amounts of DMBA can reach the bone marrow for metabolism by CYP1B1, as evidenced by the large decrease in DMBA-DNA adducts in CYP1B1-null mice relative to wild-type mice. Moreover, DMBA-DNA adduct profiles in wild-type mouse bone marrow are nearly identical to those generated with DMBA in cultured bone marrow stromal cells, where we have demonstrated expression of CYP1B1 but not CYP1A1 (17 , 36) . Although we have demonstrated DMBA induction of CYP1B1 in bone stromal cells in vitro (17 , 36) , we have yet to determine the extent to which DMBA elevates bone marrow CYP1B1 in vivo above the observed constitutive expression. Efforts are under way to identify expression of CYP1B1 by specific cell types in vivo. Metabolism by the modest levels of hepatic CYP1A1 noted in this study may largely detoxify DMBA, presumably because of the selectivity of oxidation and effective phase 2 conjugation in the liver.
Progenitor leukocytes are potential targets of DMBA metabolites.
In vivo and in vitro treatments with DMBA inhibit
lymphocyte proliferation (37
, 38)
. Cytochrome P-450 is
implicated, because the inhibitor 
-naphthoflavone alleviates this
effect in vitro (38)
. Similarly, an active
dihydrodiol epoxide metabolite of benzo(a)pyrene arrested
the cell cycle of human lymphoblasts, eventually leading to their death
(39)
. We were unable to detect a significant increase in
TUNEL-positive apoptotic bone marrow cells in wild-type mice treated
with DMBA, even when examined at time points as early as 12 h
after DMBA administration. Nevertheless, bone marrow smears of
DMBA-treated wild-type mice contained increased numbers of phagocytosed
cells, suggesting that apoptotic cells may have been quickly removed
in vivo by bone marrow macrophages. In addition, we have
shown that cultured preB cells undergo apoptosis in vitro
when these cells are incubated with wild-type bone marrow stromal
cells, which metabolize DMBA via CYP1B1. Interestingly, CYP1B1+/- mice
exhibit an intermediate bone marrow response to DMBA in
vivo, whereas bone stromal cells from these mice exhibit
intermediate activity in mediating DMBA-induced apoptosis in preB cells
in vitro. The latter observation suggests that bioactivation
of DMBA in CYP1B1+/- cells is less than in wild-type cells, probably
because of diminished CYP1B1 expression.
DMBA damage to bone marrow stromal cells in vivo may also
change their production of factors that regulate progenitor leukocyte
survival. An absence of bone marrow stromal cell growth factors,
including colony stimulating factors and interleukins 3 and 6, causes
cell death of progenitor leukocytes (40)
. Alternatively,
damage to bone marrow stromal cells may cause the release of factors,
such as tumor necrosis factor- and transforming growth factor-ß,
which can cause apoptosis of myeloid precursor cells (41)
.
Future investigations will address whether damage to stromal cells in
the bone marrow microenvironment is involved in the bone marrow
toxicity of DMBA in vivo.
The results of this investigation indicate that CYP1B1 activity has important implications for human health. Many reports have suggested that PAHs containing environmental contaminants pose a risk for human cancer development (1) , and a recent report demonstrated that human CYP1B1 activates many diverse procarcinogens, including the proximate carcinogen DMBA-3,4-dihydrodiol (42) . CYP1A1 is more effective at metabolizing DMBA than CYP1B1 but produces less of the proximate carcinogen in mice. Although DNA adducts of PAHs have been demonstrated in circulating human leukocytes, it is controversial whether they influence the development of leukemias and lymphomas (5) . A recent article substantiates the role of PAH metabolism and adduct formation in carcinogenesis by demonstrating that lung tumors arising among cigarette smokers contain mutations thought to be initiated by metabolites of benzo(a)pyrene (2 , 6) . Our work suggests that human CYP1B1 polymorphisms could pose a risk factor for the development of cancers, including those of the immune system.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by the University of
Wisconsin School of Veterinary Medicine Training Grants ES07015 and
IF32-ES05827 (to S. M. H.), by National Institute of Environmental
Health Sciences Center Grant ES09090, and by NIH Grants CA16265 (to
C. R. J.) and CA81493 (to C. J. C.). 
2 This work was performed equally in the
laboratories of C. J. C. and C. R. J.
3 To whom request for reprints should be
addressed, at Department of Pharmacology, Medical Science Center,
University of Wisconsin, 1300 University Avenue, Madison, WI 53706.
Phone: (608) 263-3128; Fax: (608) 262-1257; E-mail: jefcoate{at}facstaff.wisc.edu
4 The abbreviations used are: PAH, polycyclic
aromatic hydrocarbon; CYP, cytochrome P-450; DMBA,
7,12-dimethylbenz[a]anthracene; TUNEL, terminal
deoxynucleotidyl transferase-mediated dUTP nick end labeling; RT-PCR,
reverse transcription-PCR.
5 R. Raskin, personal communication.
6 Galvin et al., unpublished
results.
Received 12/21/99. Accepted 5/18/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Z. Yu, B. Mahadevan, C. V. Lohr, K. A. Fischer, M. A. Louderback, S. K. Krueger, C. B. Pereira, D. J. Albershardt, W. M. Baird, G. S. Bailey, et al. Indole-3-carbinol in the maternal diet provides chemoprotection for the fetus against transplacental carcinogenesis by the polycyclic aromatic hydrocarbon dibenzo[a,l]pyrene Carcinogenesis, October 1, 2006; 27(10): 2116 - 2123. [Abstract] [Full Text] [PDF]
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M. Prince, C. T. Campbell, T. A. Robertson, A. J. Wells, and H. E. Kleiner Naturally occurring coumarins inhibit 7,12-dimethylbenz[a]anthracene DNA adduct formation in mouse mammary gland Carcinogenesis, June 1, 2006; 27(6): 1204 - 1213. [Abstract] [Full Text] [PDF]
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 Z. Yu, C. V. Loehr, K. A. Fischer, M. A. Louderback, S. K. Krueger, R. H. Dashwood, N. I. Kerkvliet, C. B. Pereira, J. E. Jennings-Gee, S. T. Dance, et al. In utero Exposure of Mice to Dibenzo[a,l]Pyrene Produces Lymphoma in the Offspring: Role of the Aryl Hydrocarbon Receptor Cancer Res., January 15, 2006; 66(2): 755 - 762. [Abstract] [Full Text] [PDF]
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 J. van Grevenynghe, M. Bernard, S. Langouet, C. Le Berre, T. Fest, and O. Fardel Human CD34-Positive Hematopoietic Stem Cells Constitute Targets for Carcinogenic Polycyclic Aromatic Hydrocarbons J. Pharmacol. Exp. Ther., August 1, 2005; 314(2): 693 - 702. [Abstract] [Full Text] [PDF]
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J. Gao, F. T. Lauer, S. Dunaway, and S. W. Burchiel Cytochrome P450 1B1 Is Required for 7,12-Dimethylbenz(a)-anthracene (DMBA) Induced Spleen Cell Immunotoxicity Toxicol. Sci., July 1, 2005; 86(1): 68 - 74. [Abstract] [Full Text] [PDF]
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E. Aklillu, S. Ovrebo, I. V. Botnen, C. Otter, and M. Ingelman-Sundberg Characterization of Common CYP1B1 Variants with Different Capacity for Benzo[a]pyrene-7,8-Dihydrodiol Epoxide Formation from Benzo[a]pyrene Cancer Res., June 15, 2005; 65(12): 5105 - 5111. [Abstract] [Full Text] [PDF]
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 J. M. Ward, N. P. Nikolov, J. R. Tschetter, J. B. Kopp, F. J. Gonzalez, S. Kimura, and R. M. Siegel Progressive Glomerulonephritis and Histiocytic Sarcoma Associated with Macrophage Functional Defects in CYP1B1-Deficient Mice Toxicol Pathol, October 1, 2004; 32(6): 710 - 718. [Abstract] [PDF]
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S. Gebel, B. Gerstmayer, A. Bosio, H.-J. Haussmann, E. V. Miert, and T. Muller Gene expression profiling in respiratory tissues from rats exposed to mainstream cigarette smoke Carcinogenesis, February 1, 2004; 25(2): 169 - 178. [Abstract] [Full Text] [PDF]
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L. L. Allan, K. K. Mann, R. A. Matulka, H.-Y. Ryu, J. J. Schlezinger, and D. H. Sherr Bone Marrow Stromal-B Cell Interactions in Polycyclic Aromatic Hydrocarbon-Induced Pro/Pre-B Cell Apoptosis Toxicol. Sci., December 1, 2003; 76(2): 357 - 365. [Abstract] [Full Text] [PDF]
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T. J. Page, S. O'Brien, K. Holston, P. S. MacWilliams, C. R. Jefcoate, and C. J. Czuprynski 7,12-Dimethylbenz[a]anthracene-Induced Bone Marrow Toxicity Is p53-Dependent Toxicol. Sci., July 1, 2003; 74(1): 85 - 92. [Abstract] [Full Text] [PDF]
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 H.-Y. Ryu, K. K. Mann, J. J. Schlezinger, B. Jensen, and D. H. Sherr Environmental Chemical-Induced Pro/Pre-B Cell Apoptosis: Analysis of c-Myc, p27Kip1, and p21WAF1 Reveals a Death Pathway Distinct from Clonal Deletion J. Immunol., May 15, 2003; 170(10): 4897 - 4904. [Abstract] [Full Text] [PDF]
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T. J. Page, S. O'Brien, C. R. Jefcoate, and C. J. Czuprynski 7,12-Dimethylbenz[a]anthracene Induces Apoptosis in Murine Pre-B Cells through a Caspase-8-Dependent Pathway Mol. Pharmacol., August 1, 2002; 62(2): 313 - 319. [Abstract] [Full Text] [PDF]
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E. Aklillu, M. Oscarson, M. Hidestrand, B. Leidvik, C. Otter, and M. Ingelman-Sundberg Functional Analysis of Six Different Polymorphic CYP1B1 Enzyme Variants Found in an Ethiopian Population Mol. Pharmacol., March 1, 2002; 61(3): 586 - 594. [Abstract] [Full Text] [PDF]
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 B. Bolon and E. Galbreath Use of Genetically Engineered Mice in Drug Discovery and Development: Wielding Occam's Razor to Prune the Product Portfolio International Journal of Toxicology, January 1, 2002; 21(1): 55 - 64. [Abstract] [PDF]
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mol in
wild-type mice and 0.3 ± 0.3 
) or absence (
) of 10
µM DMBA. Apoptotic preB cells were TUNEL labeled and
enumerated by flow cytometry. A significant increase (*,
P < 0.01) in the number of apoptotic
preB cells was present in wild-type and CYP1B1+/- cocultures treated
with DMBA but not in preB cells cocultured with CYP1B1-null cells and
DMBA (P > 0.5). The data shown are the
means of a single representative experiment from three similar
experiments performed; bars, SD. C,
control; D, DMBA.