* Department of Pharmacology and Toxicology, State University of New York at Buffalo, Buffalo, New York 14214,
Department of Molecular Pharmacology and Cancer Therapeutics, Roswell Park Cancer Institute, Buffalo, New York 14263; and
Department of Molecular and Cellular Biophysics, Roswell Park Cancer Institute, Buffalo, New York 14263
Received August 6, 2003; accepted October 27, 2003
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 BaPDNA 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 BaPDNA 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 BaPDNA 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 BaPDNA 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 chemicalDNA 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 BaPDNA adduct formation in lung versus liver (Harrigan, submitted). The validation of this tissue slice model in the study of BaPDNA 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). 3HBaP (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). Waymouths medium, horse serum, and fetal calf serum were purchased from Life Technologies (Grand Island, NY).
Animals.
Male Sprague-Dawley rats (275300 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 3HBaP (6.252 µCi/rat) at concentrations of 10 or 50 mg/kg. The amount of 3HBaP 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:
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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 Waymouths 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 3HBaP (0.5 µCi/vial) at concentrations of 1, 10, or 80 µM. The amount of 3HBaP 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 TrisHCl, 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:
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Statistical analysis.
Results represent the mean ± standard deviation. Statistical analysis of tissue dosimetry studies was performed using Students 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 Dunnetts 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 BaPDNA 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 3HBaP
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 3HBaP 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 3HBaP, 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 3HBaP (µg/g tissue wet weight) at all doses and time points compared with liver slices.
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In vivo.
The concentration of radiolabeled BaP in rat liver and lung was determined following a single ip injection of 3HBaP (10 or 50 mg/kg). The amount of tissue-associated 3HBaP 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 3HBaP than lung at all times and doses examined following in vivo treatment with BaP.
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There were two major BaPDNA adducts (adducts 1 and 2) in lung slices. Adduct 1 and 2 accounted for approximately 48% and 31% of the respective BaPDNA 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 BaPDNA 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 BaPDNA adducts (data not shown). These results suggest that in vivo pretreatment with TCDD qualitatively altered the BaPDNA 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, BaPDNA 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, BaPDNA 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 BaPDNA adduct formation (Fig. 4
, compare A to C and B to D). Moreover, levels of total BaPDNA 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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The major BaPDNA 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 BaPDNA 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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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 (180 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 BaPDNA 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 BaPDNA adducts compared to non-TCDD-pretreated animals.
In rats exposed to BaP in vivo (ip injection), the accumulation of 3HBaP was significantly greater in liver tissue compared with lung tissue (Fig. 2). Conversely, in vitro treated lung slices accumulated much higher levels of 3HBaP 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 BaPDNA 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 BPDEDNA, 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 BaPDNA 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 BaPDNA 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 BaPDNA damage. Liver and lung slices catalyzed the metabolic activation of BaP, which then resulted in the formation of several BaPDNA 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 BaPDNA 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).
NOTES
1 Present address: National Institute on Aging, NIH, Baltimore, MD.
2 Present address: Pharmacy, University of Wisconsin, Madison, WI.
3 Present address: Schering-Plough Research Institute, Kenilworth, NJ.
4 To whom correspondence should be addressed at SUNY at Buffalo, Department of Pharmacology and Toxicology, 102 Farber Hall, 3435 Main St., Buffalo, NY 14214. Fax: (716) 829-2801. E mail: jolson{at}buffalo.edu.
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