sábado, 20 de marzo de 2010

DNA Adduct Formation in Precision-Cut Rat Liver and Lung Slices Exposed to Benzo[a]pyrene

ABSTRACT
Chemical-DNA adducts provide an integrated measure of exposure,
absorption, bioactivation, detoxification, and DNA repair following
exposure to a genotoxic agent. Benzo[a]pyrene (BaP), a prototypical
polycyclic aromatic hydrocarbon (PAH), can be bioactivated by
cytochrome P-450s (CYPs) and epoxide hydrolase to genotoxic
metabolites which form covalent adducts with DNA. In this study, we
utilized precision-cut rat liver and lung slices exposed to BaP to
investigate tissue-specific differences in chemical absorption and
formation of DNA adducts. To investigate the contribution of
bioactivating CYPs (such as CYP1A1 and CYP1B1) on the formation of
BaP–DNA adducts, animals were also pretreated in vivo with
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) prior to in vitro
incubation of tissue slices with BaP. Furthermore, the tissue
distribution of BaP and BaP–DNA adduct levels from in vivo studies
were compared with those from the in vitro tissue slice experiments.
The results indicate a time- and concentration-dependent increase in
tissue-associated BaP following exposure of rat liver and lung tissue
slices to BaP in vitro, with generally higher levels of BaP retained
in lung tissue. Furthermore, rat liver and lung slices metabolized BaP
to reactive intermediates that formed covalent adducts with DNA. Total
BaP–DNA adducts increased with concentration and incubation time.
Adduct levels (fmol adduct/µg DNA) in lung slices were greater than
liver at all doses. Liver slices contained one major and two minor
adducts, while lung slices contained two major and 3 minor adducts.
The tissue-specific qualitative profile of these adducts in tissue
slices was similar to that observed from in vivo studies, further
validating the use of this model. Pretreatment of animals with TCDD
prior to in vitro incubation with BaP potentiated the levels of DNA
adduct formation. TCDD pretreatment altered the adduct distribution in
lung but not in liver slices. Together, the results suggest that
tissue-specific qualitative and quantitative differences in BaP–DNA
adducts could contribute to the lung being a target tissue for BaP
carcinogenesis. Furthermore, the results validate the use of
precision-cut tissue slices incubated in dynamic organ culture as a
useful model for the study of chemical–DNA adduct formation.
Key Words: benzo(a)pyrene; DNA adduct; cytochrome P-450; TCDD; tissue slices.
Exposure to tobacco smoke has been associated with many different
types of cancer, but is responsible for up to 87% of all lung cancers
and is the greatest risk factor for developing cancer of the lung
(U.S. Department of Health and Human Services, 1989). Many of the
chemical carcinogens contained in cigarette smoke, including
benzo[a]pyrene (BaP), are members of the polycyclic aromatic
hydrocarbon (PAH) family. BaP is often used as a model compound for
PAH toxicity studies and has been shown to be a potent lung carcinogen
in the Syrian golden hamster when administered locally or by
inhalation (Thyssen et al., 1981; Wolterbeek et al., 1995). The
selective carcinogenesis of the lung following exposure to cigarette
smoke and BaP may be a consequence of many biochemical factors,
including those that affect absorption, metabolism, and DNA repair.
Considering that the tissue concentration of BaP in the rat is
elevated in the lung compared to the liver following inhalational
(Withey et al., 1993), intratracheal (Weyand and Bevan, 1986, 1987),
and intravenous (Moir et al., 1998) exposure, it suggests that BaP
absorption is greater in the lung than in other tissues.
Metabolic differences between tissues may also contribute to
variations in the rate at which BaP-mediated carcinogenesis occurs.
Several drug-metabolizing enzymes are involved in the metabolism and
activation of BaP, including cytochrome P450 1A1 (CYP1A1) (Cristou et.
al., 1995), CYP1B1 (Shimada et al., 1996) and epoxide hydrolase
(Shimada et al., 1999). Conversion of BaP to its reactive metabolite
BaP-7,-8-dihydrodiol-9,10-epoxide (BPDE) results in the rapid
formation of adducts with cellular proteins and DNA. BaP also induces
expression of CYP1A1 and CYP1B1, thus promoting its own metabolism.
Tissue-specific differences in the induction of these CYPs may also
give rise to variations in BaP-mediated carcinogenesis. The induction
of CYP1A1 and CYP1B1 is achieved through activation of the cytosolic
aryl hydrocarbon receptor (AhR) (Israel and Whitlock, 1984; Whitlock,
1999). Other pathways proposed for the metabolic activation of BaP
include the formation of radical cations catalyzed by P450 peroxidases
(Cavalieri and Rogan, 1995) and the formation of reactive and redox
active o-quinones catalyzed by dihydrodiol dehydrogenases, which are
members of the aldo-keto reductase (AKR) gene superfamily (Penning et
al., 1999).
The AhR is capable of binding a variety of environmental toxicants,
including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin), which
exhibits substantially higher affinity for the AhR compared to BaP
(Piskorska-Pliszczynska et al., 1986; Whitlock, 1993). While dioxin is
also a carcinogen, unlike BaP, dioxin resists CYP-mediated metabolism
and is generally considered nongenotoxic (Geiger and Neal, 1981). The
carcinogenic effects of TCDD may therefore be mediated through the
induction of CYP1A1 and CYP1B1, which, in the presence of genotoxic
carcinogens (including BaP), may enhance metabolic activation and
thereby increase the formation of DNA adducts.
Although much is known about the molecular mechanism of BaP-mediated
carcinogenesis, the basis for its carcinogenic activity at a given
tissue is less well understood, but depends in part on the species and
route of exposure. The purpose of this study was to analyze
tissue-specific differences in chemical absorption and formation of
DNA adducts in rat liver and lung tissues following in vivo or in
vitro treatment with BaP. We also induced levels of CYP1A1 and CYP1B1
by pretreating rats in vivo with TCDD to determine the effect of
elevated bioactivating enzymes on uptake of BaP and BaP–DNA adduct
formation in lung versus liver (Harrigan, submitted). The validation
of this tissue slice model in the study of BaP–DNA adducts, as well as
the utility of modifying drug metabolizing enzymes (both bioactivating
and detoxifying) in vivo prior to incubation of tissue slices in vitro
is discussed.
MATERIALS AND METHODS
Materials.
TCDD was a gift from Dow Chemical Co. (Midland, MI). 3H–BaP (80
Ci/mmol) was obtained from Amersham (England) and purified according
to the method described previously (DePierre et al., 1975). BaP,
gelatin, Modified Eagle Medium (MEM), HEPES, penicillin, gentamycin,
streptomycin, micrococcal nuclease (MN), spleen phosphodiesterase
(SPD), nuclease P1 (NP1), and apyrase were purchased from Sigma
Chemical Co. (St. Louis, MO). Waymouth's medium, horse serum, and
fetal calf serum were purchased from Life Technologies (Grand Island,
NY).
Animals.
Male Sprague-Dawley rats (275–300 g) were purchased from Harlan
Sprague-Dawley, Inc. (Indianapolis, IN). The animals were maintained
on a 12 h light/12 h dark cycle and received water and food ad
libitum. All animal procedures were performed in compliance with
AAALAC-approved guidelines for the humane treatment of laboratory
animals.
In Vivo Experiments
In vivo exposure.
BaP was initially dissolved in acetone, diluted with corn oil, and the
acetone evaporated under nitrogen. Rats were given a single ip
injection of BaP in corn oil (10 or 50 mg/kg) or corn oil alone
(control, 4 ml/kg) at 24 or 48 h prior to harvesting liver and lungs.
Tissues were stored at -70°C until analysis.
Tissue dosimetry.
Rats received a single ip injection containing unlabeled BaP plus
3H–BaP (6.252 µCi/rat) at concentrations of 10 or 50 mg/kg. The amount
of 3H–BaP in rat liver and lung tissue was determined at 24 or 48 h.
Tissue samples were weighed and digested overnight at 37°C in 1 N
NaOH. Samples were then acidified with 6 N HCl and diluted with
Ecoscint scintillation solution (National Diagnostics, Atlanta, GA).
BaP-derived radioactivity was measured by liquid scintillation
counting, and the concentration of BaP in liver and lung tissue was
calculated as follows:
In Vitro Experiments
In vivo pretreatment.
Rats received a single ip injection of TCDD (5 µg/kg) or corn oil
alone (control) 48 h prior to harvesting the liver and lungs. The TCDD
was dissolved in dioxane and diluted with corn oil (30 µl dioxane/ml
corn oil). Animals were pretreated with TCDD to induce expression of
dioxin-responsive CYP450s, including CYP1A1, CYP1A2, and CYP1B1.
Tissue slice preparation and incubation.
Precision-cut liver and lung slices were prepared from control and
TCDD pretreated rats essentially as described previously (Drahushuk et
al., 1996; Smith et al., 1989). Rats were anesthetized with sodium
pentobarbital (100 mg/kg). Livers were perfused with oxygenated (95%
O2:5% CO2) ice-cold Krebs-Henseleit buffer (4°C, pH 7.4) supplemented
with 20 mM glucose (KH buffer) and immediately removed and placed in
oxygenated ice-cold KH buffer. Lungs were inflated with 3% gelatin in
MEM at 37°C, as previously described (Stefaniak et al., 1992),
removed, and placed in ice-cold oxygenated KH buffer. Cylindrical
tissue cores (8 mm diameter) were prepared by slowly rotating a
sterile AcuPunch biopsy punch (Acuderm Inc., Ft. Lauderdale, FL)
through the liver and lung tissue. Precision-cut liver slices (250 µm
thick) and lung slices (450 µm thick) were prepared from individual
cylinders using a stainless steel, autoclavable version of the
Krumdieck tissue slicer (Alabama R&D Corp., Munford, AL). The slicer
was filled with oxygenated ice-cold KH Buffer, and slices were
collected in a sterile beaker. Liver (two to three slices, ~ 49 mg
total) or lung (four to five slices, ~21 mg total) slices were placed
onto a stainless steel mesh (260 µm pore size) contained within a
cylindrical stainless steel insert. Inserts were then loaded
horizontally into glass scintillation (culture) vials containing 2.5
ml of oxygenated Waymouth's medium (supplemented with 25 mM HEPES, 25
mM glucose, 5% horse serum, 5% fetal calf serum, penicillin,
gentamycin, and streptomycin). The culture vials were closed with a
cap containing a 2 mm hole for gas exchange and placed horizontally in
a custom-designed vial rotator constructed within an incubator
(University at Buffalo Instrument Shop). The dynamic organ culture
system rotated the tissue slices in and out of the medium at 1.5
rev/min in an atmosphere of 95% O2:5% CO2 at 37°C.
In vitro exposure.
Individual incubation vials were maintained for 4 or 24 h in 2.5 ml of
medium containing 1, 10, or 80 µM BaP, or medium without BaP
(control). Following 4 or 24 h of incubation, liver and lung slices
were weighed and immediately frozen at -70°C for later analysis.
Tissue dosimetry.
The concentration of BaP in liver and lung slices was determined
following incubation of the slices for 4 or 24 h in medium containing
BaP and 3H–BaP (0.5 µCi/vial) at concentrations of 1, 10, or 80 µM.
The amount of 3H–BaP in the tissue slices was determined as described
above for in vivo experiments.
DNA extraction.
High-molecular-weight DNA was isolated from rat liver and lung using
QIAGEN Genomic-tips (QIAGEN Inc., Valencia, CA) essentially as
described by the manufacturer, with the following modifications.
Samples were initially digested with RNase A for 2 h at 50°C.
Subsequently, Protease was added, and samples were digested overnight
at 50°C. Isolated DNA was resuspended in TE (10 mM Tris–HCl, pH 8.0; 1
mM EDTA) buffer, quantitated by measuring spectrophotometric
absorbance at 260 nm, and stored at -70°C until analysis.
32P-Postlabeling.
32P-postlabeling was performed essentially as described previously
(Faletto et al., 1990; Reddy and Randerath, 1986). Briefly, DNA
samples (2.5 µg) were dried and digested with 5 µg (0.6 U) MN and 5 µg
SPD for 3 h at 37°C. DNA was further digested for 1 h at 37°C with 6
µg NP1 for enrichment of adducts. A 2 µl aliquot was then removed for
HPLC quantitation of total nucleosides (Dunn et al., 1987). The
digested DNA was labeled with T4 polynucleotide kinase and 50 µCi of
[32P]-ATP (NEN, Boston, MA) for 45 min at 37°C and subsequently
treated with apyrase to convert unused [32P]-ATP to inorganic
phosphate and ADP (Gupta et al., 1982). Labeled samples were spotted
onto PEI-cellulose thin layer chromatography plates (Macherey Nagel,
Germany), and adducts were separated according to the method described
by Gupta and Randerath (1988). The chromatograms were developed using
multidirectional chromatography as described previously (Dunn et al.,
1987) using the following solvent systems: D1, 1 M NaH2PO4 pH 6.5; D2,
not performed; D3, 5.3 M lithium formate, 8.5 M urea pH 3.5; D4, 1.2 M
LiCl, 0.5 M Tris-Base, 8.5 M urea pH 8.0; D5, 1.7 M NaH2PO4 pH 6.0.
Adduct spots were detected by autoradiography using Kodak Biomax MS
film (Rochester, NY) and cut from the plates for determination of
associated radioactivity by liquid scintillation counting. DNA adducts
were quantitated as follows:
Statistical analysis.
Results represent the mean ± standard deviation. Statistical analysis
of tissue dosimetry studies was performed using Student's unpaired
t-test. Statistical analysis of DNA adduct studies was performed using
SPSS for Windows software and one-way analysis of variance (ANOVA).
Significant differences between treatment groups and control groups
were determined by Dunnett's two-sided post hoc test. A value of p <
0.05 was considered significant.
RESULTS
An in vitro precision-cut tissue slice model was utilized to examine
the tissue-specific formation of BaP–DNA adducts in rat liver and
lung. Some animals were pretreated in vivo with TCDD prior to in vitro
incubation of rat liver and lung slices with BaP. TCDD-pretreated
animals were used to examine the influence of elevated expression of
TCDD-inducible BaP bioactivating enzymes CYP1A1, CYP1A2, and CYP1B1 on
the formation of DNA adducts. The viability of liver and lung slices
incubated in dynamic organ culture with control medium or BaP
containing medium for 4 or 24 h was determined by measurement of
intracellular K+. There were no differences in intracellular K+
content in tissue slices between control and TCDD pretreated rats
(data not shown), which suggests that TCDD was not in itself
cytotoxic. Additionally, histological evaluation did not show any
overt differences in viability between control slices and tissue
slices incubated with BaP (80 µM) for 24 h (data not shown).
Uptake of 3H–BaP
In vitro.
Liver and lung slices from control and TCDD pretreated rats were
prepared and incubated for 4 or 24 h in dynamic organ culture with
radiolabeled BaP (1, 10, or 80 µM) to determine the amount of
carcinogen in each tissue. The amount of tissue-associated 3H–BaP in
liver and lung slices increased with both time (4 to 24 h) and
concentration (1 to 80 µM BaP) (Fig. 1). TCDD pretreatment did not
affect uptake of 3H–BaP, as there were no differences in uptake
between control and TCDD pretreated samples at the p < 0.05
significance level. Lung slices contained similar or greater amounts
of 3H–BaP (µg/g tissue wet weight) at all doses and time points
compared with liver slices.
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FIG. 1. Concentration of 3H–BaP in rat liver and lung slices
incubated in dynamic organ culture. Rats were treated in vivo with
TCDD (TCDD Pretreated) or corn oil (Control) 48 h prior to harvesting
the liver and lungs. Liver (A, C) and lung (B, D) slices were prepared
and incubated in dynamic organ culture for 4 (A, B) or 24 h (C, D)
with various concentrations of 3H–BaP. Tissue slices were digested
overnight, and tissue-associated radioactivity was determined by
liquid scintillation counting. The results represent the mean ± SD (n
= 4). For each media concentration, differences between Control and
TCDD Pretreated values were not statistically significant as
determined by Student's t-test. *Denotes concentrations of BaP in lung
slices that were significantly greater than the respective
concentration in liver slices.

The amount of tissue-associated 3H–BaP increased in a linear
(concentration dependent) fashion (R2 > 0.985) in liver and lung
slices incubated in dynamic organ culture with radiolabeled BaP (1,
10, or 80 µM) for 4 or 24 h (data not shown). Pretreatment with TCDD
in vivo did not affect the linearity of uptake of BaP in slices, as
there was no difference in 3H–BaP content between slices from control
and TCDD pretreated animals.
In vivo.
The concentration of radiolabeled BaP in rat liver and lung was
determined following a single ip injection of 3H–BaP (10 or 50 mg/kg).
The amount of tissue-associated 3H–BaP increased with dose (10 to 50
mg/kg), but not with time (24 to 48 h) in both liver and lung (Fig.
2). Furthermore, rat liver contained significantly greater (p < 0.05)
levels of 3H–BaP than lung at all times and doses examined following
in vivo treatment with BaP.
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FIG. 2. Concentration of 3H–BaP in rat liver and lung following in
vivo treatment with BaP. Rats were treated in vivo with 10 or 50 mg/kg
3H–BaP (6.252 µCi/rat, ip), and liver and lungs were harvested 24 or
48 h later. The tissue was digested overnight, and tissue-associated
radioactivity was determined by liquid scintillation counting. The
results represent the mean ± SD (n = 3). *Significant differences
between liver and lung within each treatment group as determined by
Student's t-test.

BaP–DNA Adduct Formation
In vitro.
The formation of BaP–DNA adducts in rat liver and lung slices was
analyzed by 32P-postlabeling. Representative autoradiographs from
32P-postlabeled DNA are shown in Figure 3. BaP–DNA adducts were
undetectable in liver and lung slices incubated in control medium
(Fig. 3A and D). Incubation with BaP resulted in the formation of
several BaP–DNA adducts in both liver and lung slices. One of the
major adducts observed in each tissue (adduct 1) comigrated with a
7R,8S,9S-trihydroxy-10S-(N2-deoxyguanosyl-3'
phosphate)7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE-N2-dG-3' P) standard
(data not shown). Pretreatment with TCDD in vivo potentiated the
levels of BaP–DNA adducts that were formed (Fig. 3, compare B with C
and E to F). Additionally, the profile of individual BaP–DNA adducts
differed between liver and lung slices (Fig. 3, compare B and C with E
and F).

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FIG. 3. Representative autoradiographs of 32P-postlabeled DNA from
rat liver and lung slices incubated with BaP in dynamic organ culture.
Liver (A, B, C) and lung (D, E, F) slices from control and
TCDD-pretreated rats were incubated in dynamic organ culture for 24 h
with various concentrations of BaP as indicated [(in vivo
pretreatment) (in vitro incubation)]. DNA was isolated from tissue
slices, and BaP–DNA adducts were determined by 32P-postlabeling.
Autoradiography was carried out for 4 h at -70°C with intensifying
screens. The origin of chromatography was at the lower left corner of
each panel. Dashed circles represent adducts that appeared following
longer exposure times. Adduct 1 comigrated with a BPDE-N2-dG-3' P
standard.

Adduct 1 predominated in liver slices and accounted for approximately
64% (control) and 58% (TCDD pretreated) of total BaP–DNA adducts
following incubation of liver slices for 24 h with 10 µM BaP (Fig.
3B). Incubation of liver slices with 80 µM BaP for 24 h increased the
relative abundance of adduct 1 to approximately 73% and 73% in control
and TCDD pretreated animals, respectively (data not shown). Therefore,
these results suggest that TCDD pretreatment in vivo did not
qualitatively alter the BaP–DNA adduct profile in liver slices
incubated with BaP in vitro.
There were two major BaP–DNA adducts (adducts 1 and 2) in lung slices.
Adduct 1 and 2 accounted for approximately 48% and 31% of the
respective BaP–DNA adducts in lung slices incubated with 10 µM BaP for
24 h (Fig. 3E). Incubation of lung slices with a higher concentration
of BaP (80 µM, 24 h) produced similar results, where adducts 1 and 2
constituted 45% and 31%, of total BaP–DNA adducts, respectively (data
not shown). In lung slices from animals pretreated in vivo with TCDD
and incubated for 24 h with 10 µM BaP, adduct 1 accounted for
approximately 37% of the total detectable adducts, while adduct 2 made
up 55% of the total (Fig. 3F). Thus, TCDD pretreatment increased the
abundance of adduct 2 relative to adduct 1. Incubation of lung slices,
from TCDD pretreated rats, with 80 µM BaP produced a similar adduct
profile, where adduct 2 and adduct 1, respectively, accounted for 54%
and 38% of the total BaP–DNA adducts (data not shown). These results
suggest that in vivo pretreatment with TCDD qualitatively altered the
BaP–DNA adduct profile in rat lung slices, increasing the level of
adduct 2 relative to adduct 1.
Total adduct levels for rat liver and lung slices incubated in dynamic
organ culture with BaP are shown in Figure 4. After 4 h of incubation,
BaP–DNA adducts increased in a concentration-dependent manner in
animals pretreated with TCDD in vivo, but were not detectable in
animals pretreated with corn oil (Fig. 4A and B). However, following
24 h of incubation with BaP in dynamic organ culture, BaP–DNA adducts
increased in a concentration-dependent manner in both control and TCDD
pretreated animals (Fig. 4, C and D). Both liver and lung slices
showed a time-dependent increase in total BaP–DNA adduct formation
(Fig. 4, compare A to C and B to D). Moreover, levels of total BaP–DNA
adducts were greater in lung slices compared to liver at each dose
examined (Fig. 4, compare A with B and C with D).
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FIG. 4. BaP–DNA adduct levels in rat liver and lung slices
incubated with BaP in dynamic organ culture. Liver (A, C) and lung (B,
D) slices from control and TCDD-pretreated rats were incubated in
dynamic organ culture for 4 (A, B) or 24 h (C, D) with various
concentrations of BaP. DNA was isolated from tissue slices, and
BaP–DNA adducts were quantitated by 32P-postlabeling. The results
represent the mean ± SD (n = 4–6). *Significant differences between
treatment groups and their respective controls (0 µM BaP) using ANOVA
with Dunnett's post hoc test at p < 0.05.

In vivo.
BaP–DNA adduct formation was also examined by 32P-postlabeling in rats
receiving a single ip injection of corn oil (control) or BaP (10 or 50
mg/kg) at 24 or 48 h prior to harvesting liver and lungs.
Representative autoradiographs from those studies are shown in Figure
5. Treatment of rats with BaP in vivo generally resulted in lower DNA
adduct levels than treatment of tissue slices with BaP in vitro, as
autoradiography required 24 h exposure time for in vivo treatment
studies, compared to 4 h for the tissue slice studies. Animals
receiving corn oil alone did not show any detectable BaP–DNA adducts
(Fig. 5A and D). However, in vivo treatment with BaP (10 and 50 mg/kg)
resulted in the formation of BaP–DNA adducts in both liver and lung
(Fig. 5B, C, E, and F). The individual BaP–DNA adduct profile differed
between liver and lung, but the overall profile for each tissue was
similar to that seen from the in vitro tissue slice studies (compare
Fig. 5 with Fig. 3).

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FIG. 5. Representative autoradiographs of 32P-postlabeled DNA from
rat liver and lung treated in vivo with BaP. Liver and lung tissue was
obtained from rats 24 h following treatment with corn oil (Control) or
BaP (10 or 50 mg/kg BaP, ip). DNA was isolated from rat liver (A, B,
C) and lung (D, E, F), and BaP–DNA adducts were determined by
32P-postlabeling. Autoradiography was carried out for 24 h at -70°C
with intensifying screens. The origin of chromatography was at the
lower left corner of each panel. Adduct 1 comigrated with a
BPDE-N2-dG-3' P standard.

Adducts 1 and 3 predominated in liver tissue following in vivo
treatment of animals with BaP. Adduct 1 comprised approximately 50%
(10 mg/kg, Fig. 5B) and 55% (50 mg/kg, Fig. 5C) of total BaP–DNA
adducts after 24 h. Adduct 3 comprised 44 and 41% of total adducts
following treatment with 10 (Fig. 5B) and 50 (Fig. 5C) mg/kg BaP,
respectively. Similar results were observed at 48 h following in vivo
treatment with 50 mg/kg BaP (data not shown).
The major BaP–DNA adducts in lung following in vivo treatment with BaP
were adducts 1 and 2. At 24 h, adduct 1 accounted for approximately
48% and 44% of adducts in rats treated in vivo with 10 (Fig. 5E) or 50
(Fig. 5F) mg/kg BaP, respectively. Adduct 2 comprised 35% (10 mg/kg,
Fig. 5E) and 42% (50 mg/kg, Fig. 5F) of total BaP–DNA adducts. Similar
results were seen at 48 h (data not shown).
Total adduct levels in liver and lung increased in both a time- and
concentration-dependent manner in rats treated in vivo with BaP (Fig.
6). Total adduct levels were similar between liver and lung at 24 h
(10 and 50 mg/kg), but were greater in the liver at 48 h (50 mg/kg).
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FIG. 6. BaP–DNA adduct levels in rat liver and lung following in
vivo treatment with BaP. Liver and lung tissue was obtained from rats
at 24 or 48 h following treatment with corn oil (control) or BaP (10
or 50 mg/kg, ip). DNA was isolated from tissues, and BaP–DNA adducts
were quantitated by 32P-postlabeling. The results represent the mean ±
SD (n = 4). *Significant differences between treatment groups and
their respective controls using ANOVA and Dunnett's post hoc test at p
< 0.05.

DISCUSSION
Our laboratory previously utilized precision-cut liver slices (rat and
human) incubated in dynamic organ culture as an in vitro model to
assess the modulation of CYP1A1 and CYP1A2 by TCDD (Drahushuk et al.,
1996, 1998, 1999). The purpose of this study was to extend the use of
this model to investigate tissue specific differences in covalent DNA
damage caused by the ubiquitous environmental carcinogen BaP.
The results from this study demonstrate that incubation of rat liver
and lung slices with BaP (1–80 µM) did not result in cytotoxicity.
Tissue-associated BaP increased in a time- and concentration-dependent
manner, and lung slices generally contained greater levels of BaP
compared to liver slices (Fig. 1). Consistent with this observation,
DNA adduct levels were higher in lung slices compared to liver. Total
BaP–DNA adducts increased in a time- and concentration-dependent
manner in both rat liver and lung slices following incubation with BaP
(Fig. 4). In addition, pretreatment of animals in vivo with TCDD
resulted in an induction of CYP1A1 and CYP1B1 (Harrigan et al.,
submitted), which correlated with greater levels of BaP–DNA adducts
compared to non-TCDD-pretreated animals.
In rats exposed to BaP in vivo (ip injection), the accumulation of
3H–BaP was significantly greater in liver tissue compared with lung
tissue (Fig. 2). Conversely, in vitro treated lung slices accumulated
much higher levels of 3H–BaP than liver slices (Fig. 1). These in
vitro findings are supported by additional studies showing a selective
accumulation of BaP in lung compared to liver following inhalational
(Withey et al., 1993), intratracheal (Weyand and Bevan, 1986, 1987),
and intravenous (Moir et al., 1998) exposure. Bevan and Weyand (1988)
demonstrated that the disposition of BaP following the initial route
of administration is affected by considerable recycling of BaP from
the blood back to the lungs. Furthermore, it was noted that the lung
is the only organ to receive the total cardiac output and is the organ
of first exposure to xenobiotics in studies using inhalational,
intramuscular, intravenous, and subcutaneous routes of administration
(Roth and Vinegar, 1990). Therefore, the use of rat liver and lung
slices incubated in dynamic organ culture with BaP is an appropriate
model system, because the level of tissue-associated BaP was generally
greater in lung slices compared with liver, which correlates well with
the tissue distribution of BaP following several different in vivo
routes of exposure.
The adduct profiles for in vitro treated tissue slices (Fig. 3) were
similar to those observed in tissues from rats treated in vivo with
BaP (Fig. 5). In addition, the adduct profile of in vitro treated lung
slices generally mirrored that observed in lung tissue from in vivo
treated rats, where adduct 1 and 2 each represented approximately 40%
of the total BaP–DNA adducts detected in lung tissue. Two major DNA
adducts were also observed in lung tissue of male CD rats treated with
BaP in vivo (Ross et al., 1990). While one of these adducts was
characterized as BPDE–DNA, the second adduct was novel and accounted
for approximately 40% of the total adducts. Fang et al.(2001) later
identified this second adduct as the reaction of 9-OH-BaP-4,5-oxide
with the N2 position of dG. As this 9-OH-BaP derived DNA adduct
predominates in rat lung, and 9-OH-BaP has been found to be mutagenic,
this DNA adduct may also be associated with lung cancer.
Pretreatment of animals in vivo with TCDD potentiated the formation of
BaP–DNA adducts in rat liver and lung slices (Fig. 4). CYP1A1 and
CYP1B1 mRNA levels were also maximally induced following TCDD
pretreatment (Harrigan et al., submitted), suggesting that increased
levels of bioactivating enzymes result in greater formation of BaP–DNA
adducts. Conversely, resistant fish, which showed poor induction of
enzymes regulated by the AhR, had lower levels of adducts following
exposure to BaP (Nacci et al., 2002). In addition, BaP treatment of
AhR-deficient mice was not associated with the induction of CYP1A1 or
the formation of skin tumors. In addition, treatment of AhR-deficient
mice with BaP did not induce expression of CYP1A1 and did not produce
skin tumors (Shimizu et al., 2000). Altogether, these results suggest
that prevention of CYP1A1 and CYP1B1 induction following PAH exposure
may provide protection from the long-term consequences of DNA adduct
formation, such as cancer.
The results from this study provide evidence that the incubation of
rat liver and lung slices in dynamic organ culture with BaP is a valid
and useful in vitro model for the investigation of BaP–DNA damage.
Liver and lung slices catalyzed the metabolic activation of BaP, which
then resulted in the formation of several BaP–DNA adducts. The profile
of these adducts was similar to that observed from in vivo studies,
further validating the use of this model. It will be important to
identify the remaining BaP–DNA adducts in future studies, as
DNA-adduct profiles differed between liver and lung tissue. The
structural identification of lung-specific BaP adducts may provide
useful insights into the tissue specificity of lung carcinogenesis.
The further validation of the in vitro model utilizing precision-cut
tissue slices in dynamic organ culture in this study supports the use
of this model to reduce the number of animals required for in vivo
studies. This model can also be applied to the study of human tissues,
including human lung, and may serve as an important tool to address
critical factors associated with smoking and lung cancer risk. This
model can assist studies of carcinogen metabolism and binding to DNA
in human lung, the effects of cigarette smoke on DNA repair and adduct
persistence, the relationship between specific carcinogens and
mutations in critical genes, and the sequence of gene changes that
lead to lung cancer (Hecht, 1999).
Hecho por : Willson A Mendoza C.
C.I:16.959.604
CRF
FUENTE: http://toxsci.oxfordjournals.org/cgi/content/full/77/2/307

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