Low Levels of Expression of Cytochromes P-450 in Normal and Cancerous Fetal Pancreatic Tissues of Hamsters Treated with NNK and/or Ethanol

Liyan Zhang*,1, Diann L. Weddle{dagger},2, Paul E. Thomas{ddagger}, Beiyao Zheng§, Andre Castonguay, Hildegard M. Schuller{dagger} and Mark Steven Miller*,3

* Departments of Cancer Biology and § Public Health Sciences, Comprehensive Cancer Center, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157; {dagger} Experimental Oncology Laboratory, University of Tennessee College of Veterinary Medicine, Knoxville, Tennessee 37901; {ddagger} Department of Chemical Biology, College of Pharmacy, Rutgers University, Piscataway, New Jersey 08855; and Laboratory of Cancer Etiology and Chemoprevention, Faculty of Pharmacy, Laval University, Québec, Canada

Received February 16, 2000; accepted May 3, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Previous studies from this laboratory have demonstrated that administration of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) to pregnant hamsters results in tumors in the offspring. Whereas treatment with NNK alone caused mainly tumors in the respiratory tract of the treated offspring, cotreatment with ethanol (EtOH) and NNK shifted the site of tumor formation to the pancreas. In order to determine potential mechanisms for the cocarcinogenic effects of EtOH, the levels of NNK metabolites and expression of various CYPs implicated in the metabolic activation of NNK were determined in fetal liver and pancreas. NNK and its metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), were detected at low and variable levels in the fetal liver and pancreas, with an NNAL to NNK ratio greater than 20 in both organs. EtOH had no effect on the amount of metabolites found in either organ. Results obtained with the fetal liver samples, which served as a positive control, correlated very well with our previous studies demonstrating low levels of expression of several CYP isozymes at both the protein and RNA level. Western blot analysis showed low but detectable levels of CYP1A1, barely detectable levels of CYP2E1, and an absence of CYP1A2 and 2B family members in the fetal pancreas. RNA transcripts were undetectable by ribonuclease protection in the fetal pancreas, although readily seen in fetal liver samples. Treatment with NNK, EtOH, or both NNK and EtOH had small and variable effects on the levels of metabolism of NNK and expression of the isozymes. These findings suggest that alternative mechanisms may be responsible for transplacentally induced tumors in this model system.

Key Words: cytochromes P-450; pancreatic cancer; ethanol; NNK; fetus; transplacental carcinogenesis; tobacco-specific nitrosamines.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Research conducted in animal models has shown that the developing fetus displays a higher sensitivity toward certain chemical and physical carcinogens than does the adult organism (Miller et al., 1990Go; Miller, 1994Go; Rice, 1979Go). In addition, epidemiological studies have demonstrated elevated incidences of childhood cancers in the offspring of individuals exposed to suspected environmental carcinogens (Bunin et al., 1993Go; Hansen et al., 1992Go; John et al., 1991Go; Perera et al., 1996Go; Sandler et al., 1985Go; Stjernfeldt et al., 1986Go). These results suggest that exposure of pregnant women to environmental toxicants may place the embryo and fetus at higher risk for the development of cancer. Despite this differential sensitivity, few studies have examined the mechanisms of cancer causation and toxic responses to environmental chemicals during embryonic and fetal development. Previous studies from this laboratory have demonstrated that treatment of pregnant mice with the polycyclic aromatic hydrocarbon (PAH) 3-methylcholanthrene resulted in the formation of lung and liver tumors in the transplacentally treated offspring 1 year after birth (Wessner et al., 1996Go). Similar to the results obtained in adult murine models and observed in human lung adenocarcinomas, the lung tumors exhibited mutations in the Ki-ras gene that appeared to be associated with the initiation phase of the carcinogenic process (Leone-Kabler et al., 1997Go; Wessner et al., 1996Go). In addition, the transplacentally induced tumors also showed alterations in the expression and/or structure of several tumor suppressor genes implicated in human lung cancer (Gressani et al., 1998Go, 1999Go; Rollins et al., 1998Go). These results suggest that tumor initiation can occur during the period of fetal development.

Pancreatic cancer is the 4th leading cause of cancer-related deaths in the United States, with only about 4% of patients surviving 5 years beyond the initial diagnosis (Landis et al., 1999Go). Despite the dismal prognosis associated with this disease, the etiology and molecular pathogenesis of pancreatic cancer is still poorly understood. As demonstrated for the induction of lung and liver tumors in mice following in utero exposure to PAHs, treatment of pregnant hamsters with N-nitrosobis(2-oxopropyl)amine (BOP) caused ductular tumors in the exocrine pancreas in the exposed offspring within 1 year after birth (Pour, 1986Go). It was subsequently shown that BOP-induced tumors contained mutations in the Ki-ras and p53 genes (Cerny et al., 1990Go, 1992Go; Chang et al., 1995Go; Fujii et al., 1990Go; Rozenblum et al., 1997Go; Sugio et al., 1996Go) similar to the lesions observed in a majority of human pancreatic tumors (Almoguera et al., 1988Go; Hruban et al., 1993Go; Pellegata et al., 1994Go; Scarpa et al., 1993Go). Tobacco smoke has been implicated as a potential risk factor for pancreatic cancer, and recent studies have also suggested that ethanol (EtOH) may act as a cocarcinogen with the constituents of cigarette smoke to enhance tumor formation (Clavel et al., 1989Go; Durbec et al., 1983Go; Farrow and Davis, 1990Go; Ghadirian et al., 1991Go; Heuch et al., 1983Go; Malats et al., 1997Go; Silverman et al., 1995Go; Zheng et al., 1993Go). Among the tobacco-specific nitrosamines, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) has been shown to be a particularly potent carcinogen in animal models, with 70 to 100% of treated animals developing lung tumors (Belinsky et al., 1989Go; Hecht, et al. 1986Go; Hoffmann et al., 1981Go).

In addition to its effects in adult rodents, administration of NNK to pregnant hamsters (Correa et al., 1990Go) and mice (Anderson et al., 1989Go) caused a variety of tumors in the transplacentally exposed offspring. Whereas mice treated in utero showed mostly lung and liver tumors, exposed hamsters developed tumors in the pancreas, liver, respiratory tract, and adrenal glands as well. These findings were consistent with studies demonstrating the placental transfer, metabolism, and genotoxicity of NNK in the pregnant hamster and developing fetus (Alaoui-Jamali et al., 1989Go; Jorquera et al., 1992aGo; Rossignol et al., 1989Go). Interestingly, cotreatment of pregnant hamsters with EtOH and NNK shifted the primary site of tumor induction from the respiratory tract to the exocrine pancreas (Schuller et al., 1993Go). Although EtOH treatment resulted in an increase in the overall rate of NNK metabolism in fetal lung and liver microsomes (Jorquera et al., 1992bGo), little effect on the levels of CYP2E- and 2B-related4 family members were seen by Western and Northern blot analyses in these tissues (Miller et al., 1992Go).

Several studies have shown that a number of CYP isozymes metabolize NNK to reactive intermediates capable of binding to and damaging DNA, including CYP1A2, 2B1, and members of the 2A and 3A gene families (Guo et al., 1991Go; reviewed in Hecht, 1998Go). The presence of elevated levels of DNA adducts in human pancreatic cancer tissue, relative to normal organ donors, has been demonstrated with the P32-postlabeling assay (Wang et al., 1998Go), suggesting that metabolic activation of environmental carcinogens by pancreatic enzymes could lead to DNA damage and the observed mutations in Ki-ras and p53 described above (Almoguera et al., 1988Go; Hruban et al., 1993Go; Pellegata et al., 1994Go; Scarpa et al., 1993Go). Along these lines, Anderson et al. (1997) demonstrated that aromatic amines were metabolically activated to DNA-binding species by human pancreatic tissue. However, the metabolism of several other chemical carcinogens and CYP substrates, including NNK, was not observed. In addition, these investigators were unable to detect CYP1A2, 2A6, 2D6, 2E1, 3A3, and members of the 2C family in their pancreatic tissue preparations by immunoblotting.

Both CYP2E1 and 2B1/2 are induced by treatment with EtOH (Ardies et al., 1987Go; Koop et al., 1985Go; Kubota et al., 1988Go; Song et al., 1986Go), and the elevated incidence of pancreatic tumors in EtOH-exposed animals suggests that induction of EtOH-inducible CYPs and concomitant increase in NNK metabolism may be partly responsible for this effect. Previous studies using the pharmacogenetic mouse model have clearly demonstrated that the high sensitivity of the developing fetus to PAH-mediated lung carcinogenesis is due, in part, to the unique biochemistry of the fetus in terms of its metabolic response to environmental carcinogens (Miller et al., 1990Go, 1994, 1996Go). Because of the conflicting results obtained with adult animal and human pancreatic tissue, we have examined fetal pancreatic tissues in order to determine the role of CYP enzymes in mediating cancer initiation in the developing fetal pancreas.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials
NNK (99% pure by thin layer chromatography) and [5-3H]NNK (4.66 Ci/mmol; 99% pure by high-performance liquid chromatography [HPLC]) were supplied by Chemsyn Science Laboratory (Lenexa, KS). The synthesis of NNK metabolites used as standards in HPLC analysis has been reported (Hecht et al., 1980Go). The polyclonal rabbit anti-rat CYP1A2 and goat anti-rat CYP2B1 sera were obtained from Daiichi Pure Chemicals Co., Ltd. (Tokyo, Japan), and the rabbit anti-actin and horseradish peroxidase labeled rabbit anti-goat IgG from Sigma-Aldrich, Inc. (St. Louis, MO). Polyclonal rabbit anti-rat CYP2E1 was prepared as previously described (Thomas et al., 1987Go); horseradish peroxidase labeled goat anti-rabbit IgG was from Bio-Rad Laboratories (Hercules, CA). The SuperSignal® chemiluminescence substrate and ImmunoPure® IgG elution buffer were purchased from Pierce (Rockford, IL). The QIAquick PCR purification kit and QIAamp tissue kit were from QIAGEN Inc. (Valencia, CA). The MAXIscriptTM in vitro transcription kit, RPA IIITM ribonuclease protection assay kit, BrightStarTM Psoralen-Biotin nonisotopic labeling kit, BrightStarTM BioDetect nonisotopic detection kit, BrightStarTM BiotinylatedRNA CenturyTM size marker, and BrightStar-PlusTM positively charged nylon membrane were purchased from Ambion Inc. (Austin, TX).

Animal Treatment
For analysis of NNK metabolite levels, two groups of three Syrian golden hamsters were given either normal drinking water or 10% EtOH (v/v) in the drinking water from days 5 through 15 of gestation. Both groups of hamsters were instilled intratracheally with NNK dissolved in sterile water (100 mg/kg of body weight, 4.8 mCi/mmol, 100 mg/ml) on day 15 of gestation. Two hours after administration of NNK, the mothers were heavily anesthetized with 1 ml/kg of a mixture consisting of 900 mg ketamine and 100 mg xylazine in 10 ml. The hamsters were then euthanized by exsanguination. The fetuses were removed, placed on ice, and fetal liver and pancreatic tissues and maternal liver were removed and frozen until assayed for NNK and NNK metabolites. Previous studies have shown that treatment of pregnant hamsters with EtOH alone caused pancreatitis but did not induce pancreatic tumors in the offspring (Correa et al., 1990Go; Schuller et al., 1993Go).

For Western and ribonuclease protection assays, pregnant hamsters were exposed to 10% EtOH in the drinking water from gestation days 5 to 15. On day 15, NNK was administered by intratracheal instillation at a dose of 50 mg/kg. The treated hamsters were divided into four groups consisting of three pregnant mothers per group: control (no treatment), treatment with 10% EtOH, treatment with NNK, and cotreatment with 10% EtOH and NNK. Four hours later the mothers were heavily anesthetized and euthanized as described above. The fetuses were removed, placed on ice, and liver and pancreatic tissues processed as described below; fetal tissues from the same litters were pooled. All animal studies were approved by the University of Tennessee Institutional Animal Care and Use Committee and followed NIH and AVA guidelines for the care, use, and euthanasia of rodents.

Extraction and HPLC Analysis of NNK Metabolites
All procedures were carried out on ice. Tissue samples (0.1 g of fetal tissues or 1.0 g of maternal tissues) were homogenized in 0.1 M HCl (5 ml/g) using a Pyrex potter and precipitated with 3 volumes of methanol. After centrifugation and filtration, supernatants were evaporated to dryness under nitrogen and redissolved in the HPLC mobile phase. They were analyzed for NNK and its metabolites with a Sperisorb ODS 2 column. The elution program consisted of solvent A for 10 min followed by a linear gradient to 60% solvent B in 60 min at a flow rate of 1 ml/min. Solvent A was 60 ml of 1 M acetic acid and 24 ml of 1 M sodium hydroxide diluted to 1 liter. The pH was adjusted to 6.0 with 1 M sodium hydroxide. Solvent B was methanol:water (1:1). Aliquots (100 µl) of extracts and 7 µl of NNK metabolite standards were coinjected and eluted with a sodium acetate buffer (pH 6.0) and methanol mobile phase, as described previously (Jorquera et al., 1992bGo). The elution was monitored at 254 nm and 1-ml fractions were collected. Five milliliters of Scintisafe Plus (Fisher Scientific, Montreal, Canada) were added to each fraction, and radioactivity was measured by liquid scintillation spectroscopy. Recovery of total radioactivity during HPLC analysis was > 70%. Peaks less than twice the background level were considered insignificant. Data were expressed as the ratio of each NNK metabolite to the sum of NNK plus all metabolites in order to reduce the interindividual variability in total level of radioactivity.

Western Immunoblot Analysis
Liver and pancreas tissues were homogenized for 10–15 s in 5 volumes of 0.1 mM potassium phosphate buffer, pH 7.25, with a Biospec Tissue Tearor. The homogenate was centrifuged at 800 x g for 10 min at 4°C and protein content of the supernatant was measured by the method of Bradford (1976) using bovine serum albumin (BSA) as a standard. Twenty five micrograms of liver supernatant or 100 µg of pancreatic supernatant were then separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred electrophoretically to nitrocellulose filters using 25 mM Tris/192 mM glycine buffer (pH 8.2)/20% methanol according to the procedure of Towbin et al. (1979). For immunostaining, the blotted nitrocellulose filters were blocked for 2 h in 5% fat-free dried milk in 20 mM Tris and 500 mM NaCl, pH 7.5 (TBS), then incubated with primary antibody (1:2000) or respective preimmune serum (1:2000) for 2 h in TBS containing 0.05% Tween-20 (TTBS), followed by peroxidase conjugated secondary antibody (1:4000) for 1 h in TTBS at room temperature. The blots were washed thoroughly with TTBS after removing the primary and secondary antibodies, respectively. After initial staining, the filters were stripped by either incubating in 2% SDS/62.5 mM Tris–HCl, pH 6.8/100 mM ß-mercaptoethanol for 30 min at 65–70°C or in ImmunoPure® IgG elution buffer for 2 h at room temperature and restained with each of the indicated antibodies. The immunoreactive protein was visualized by chemiluminescence on Kodak X-Omat film according to the manufacturer's instructions, and the bands were quantitated by densitometry using a EPSON® ES-1200C image scanner and the ImageQUANTTM software program. Each immunoreactive protein quantitation was standardized to the ß-actin signal.

RNA Isolation
Total cellular RNA was isolated as described previously (Miller et al., 1992Go) by the method of MacDonald et al. (1987). Briefly, liver and pancreatic tissues were homogenized in 8 ml of ice-cold 4 M guanidine isothiocyanate/0.1 M Tris–HCl (pH 7.5)/1% 2-mercaptoethanol solution, and the homogenates were centrifuged on a cushion consisting of 3.1 ml of 6.1 M CsCl/25 mM Na acetate (pH 5.2)/10 mM EDTA in a Beckman SW41 rotor at 110,000 x g for 18–20 h at 20°C. The RNA pellets were redissolved in 0.5 ml of diethylpyrocarbonate (DEPC)-treated water and reprecipitated from a 70% EtOH/0.3 M sodium acetate (pH 5.2) solution by centrifugation. Following an additional wash with 70% EtOH, RNA was redissolved in DEPC-treated water and the concentration was determined by measuring UV absorbance at 260 nm.

Ribonuclease Protection Assay
Preparation of DNA templates.
Syrian golden hamster genomic DNA was isolated from the liver using the QIAamp tissue kit according to the manufacturer's instructions. CYP1A2, 2B1/2, and 2E1 DNA fragments were then amplified by using the following primer pairs:

5'-GGTAGAATCAGTGGCTAACG-3'(ASF3) and

5'-GGCGATGGGATTTACAACC-3' (AAS3) for CYP1A2;

5'-AGTCTCATGATGCGAGGAGCCCAGTATCTTGCT-3' (2BSF) and

5'-GCATGAAGGAATTGAGGAG-3' (2BAS) for CYP2B1/2;

5'-AGTCTCATGATGCGACCCTTGGCTATATAA-3' (2E1SF) and

5'-GCTCTATCTGCCTGG-3' (2E1AS) for CYP2E1.

Each antisense primer contained an SP6 promoter (underlined). Primers for 1A2 and 2E1 are based on hamster-specific sequences, whereas 2B1/2 primers are based on the rat sequence. 2B1/2 and 2E1 5' primers contain a 15-base nonhomologous region shown in italics. All primers are based on exon sequences except the 1A2 3' primer, which is based on the intron sequence. The amplification cycles for CYP1A2 consisted of an initial denaturation at 94°C for 4 min, 50 cycles of denaturation at 94°C for 1 min, annealing at 53°C for 1 min, and elongation at 74°C for 1 min, followed by an elongation step for 4 min at 72°C. For CYP2B1/2, the amplification conditions were initial denaturation at 94°C for 4 min, 50 cycles of 1 min at 94°C, 1 min at 55°C, and 45 s at 72°C followed by a final elongation step for 4 min at 72°C. For the amplification of CYP2E1, the PCR reaction mixtures were denatured at 94°C for 4 min, then subjected to 45 cycles of 1 min at 94°C, 1 min at 48°C, and 45 s at 72°C, followed by an elongation step at 72°C for 4 min. PCR products of 311, 202, and 236 bp were produced for CYP1A2, 2B1/2, and 2E1, respectively, and were confirmed by direct automated sequencing.

Transcription and purification of RNA probes.
RNA probes were transcribed directly from the PCR products according to the manufacturer's instructions with the MAXIscriptTM in vitro transcription kit. The transcription reaction contained 5 µl of PCR product, 1x transcription buffer, 0.5 mM each of ATP, CTP, GTP, and UTP, 20 U of SP6 RNA polymerase and 10 U of ribonuclease inhibitor in a total volume of 20 µl. The reaction mixture was incubated for 2 h at 37°C. Following treatment with DNase I for 15 min at 37°C, the entire reaction was electrophoresed on a 0.75 mm thick 5% polyacrylamide/8 M urea gel and the full-length transcripts were excised from the gel under 245 nm UV shadowing in a dark environment. The gel slice was incubated with probe elution buffer (0.5 M ammonium acetate/1 mM EDTA/0.2% SDS) at 37°C overnight, followed by precipitation of eluted RNA with the addition of 3 volumes of 100% EtOH. The RNA pellet was redissolved in TE buffer. The RNA transcripts for CYP1A2, 2B1/2, and 2E1 were 294, 185, and 219 bp, respectively.

Labeling of RNA probes.
Gel-purified RNA transcripts were labeled with psoralen-biotin based on the instruction manual provided by the manufacturer. Briefly, 10 µl of RNA solution was mixed with 1 µl of psoralen-biotin reagent and irradiated in a flat-bottomed, untreated microtiter plate with 365 nm UV light for 45 min, followed by extraction twice with water-saturated n-butanol. The labeled probe was stored at –70°C until use.

Hybridization of probe and sample RNA and separation of protected fragment.
The hybridization of the labeled RNA probe with sample RNA was carried out using the RPA IIITM kit according to the manufacturer's instructions. Thirty micrograms of total RNA and probe were coprecipitated and hybridized in 10 µl of hybridization buffer at 42°C for 18–24 h, followed by digestion with RNase A/RNase T1 (1:50 dilution for CYP1A2; 1:100 dilution for CYP2B1/2 and 2E1) at 37°C for 30 min. The protected fragment—256, 167, and 203 bp long for CYP1A2, 2B1/2, and 2E1, respectively —was then precipitated and separated on a 0.75 mm thick 5% acrylamide/8M urea gel by electrophoresis at a constant 250 V in 1 x TBE buffer (100 mM Tris borate/90 mM EDTA) for 1.5 h. The gel was electroblotted onto a positively charged nylon membrane at a constant 400 mA in 0.5 x TBE for 30 min. In addition, the mouse ß-actin probe transcribed from pTRI-actin (contained in the Maxiscript kit) was used as loading control, which was hybridized to fetal liver (10 µg) and fetal pancreas (5 µg) RNA and digested with RNase T1 only.

Detection of hybridized probe.
Nonisotopic detection of biotinylated RNA probe was carried out using the BrightStarTM Biodetect kitTM based on the instruction manual. Briefly, the blotted nylon membrane was blocked in 1x blocking buffer for 30 min and incubated with 1:10,000 diluted streptavidin-alkaline phosphatase conjugate solution for 30 min, followed by thorough washing once with 1x blocking buffer for 15 min and 3 times with 1x wash buffer for 15 min. The membrane was then incubated with the chemiluminescent CDP-StarTM reagent for 5 min and exposed to X-ray film to visualize the image. The signal was quantitated by densitometry as described for the Western blot analysis.

Statistics
Data for the HPLC analysis of metabolites were compared by a complete or incomplete (according to the case) block design ANOVA. When a factor was significant, Bonferonni's multiple comparisons (Miller, 1981Go) were carried out to determine which levels of the factor differed from the others. The extent of {alpha}-carbon hydroxylation is presented as the sum of the molar recovery of metabolites 12, 13, 14, and 15, expressed as a percentage of the total recovery of NNK, NNAL, and all metabolites; the extent of N-oxidation is presented as the sum of the molar recovery of metabolites 1 and 2 expressed as a percentage of the total recovery of NNK, NNAL, and all metabolites.

The data for Western blotting and ribonuclease protection assays was generated by a two-way factorial design with two levels (absent or present) for each of the factors, NNK and EtOH. The dependent variables were the individual isozymes. A separate analysis of variance was done for each dependent variable. Testing was initially done to determine if there was an interaction between NNK and EtOH; no significant interaction was detected. The interaction was then taken out of the model and the data tested for the main effects, using square root transformations to stabilize the variance and normalize the response distribution.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Determination of NNK Metabolism in Fetal Liver and Pancreas
The metabolism of NNK is illustrated in Figure 1Go. Carbonyl reduction of NNK results in the formation of NNAL. This reaction has been shown to be reversible (Hecht, 1998Go). Activation of NNK by {alpha}-carbon hydroxylation gives three electrophilic and mutagenic intermediates 7, 9, and 11, which are further metabolized to 12, 13, 14, and 15. Detoxification of NNK and NNAL by N-oxidation gives metabolites 1 and 2. The results shown in Tables 1 and 2GoGo demonstrated that treatment with EtOH had little effect on the relative abundance of NNK and its metabolites in maternal liver or fetal liver and pancreatic tissues; maternal liver samples were included as a comparison to levels of fetal metabolism. In the last column of Table 1Go, the sum of all metabolites formed by {alpha}-carbon hydroxylation and N-oxidation were expressed in picomoles per milligram of tissue. Although it appears that treatment with EtOH may result in a 5-fold increase in the amount of NNK metabolites reaching the fetal compartment compared with treatment with NNK alone, there was a large amount of interanimal variation within hamsters from the same treatment group. For example, in maternal liver treated with NNK alone, the values obtained ranged from 3 to 97 pmol per mg of tissue. This likely reflects the variability of the dosage received by the hamsters following intragastric administration of the NNK. Thus, expression of the data as the ratio of each metabolite or metabolite pathway over all metabolites + NNK leads to a more conservative interpretation of the data. Comparing metabolite ratios, slight trends were noted indicating that EtOH might inhibit the metabolism of NNK in maternal liver, whereas the six NNK metabolites appeared to be slightly less abundant in fetal liver of NNK-treated hamsters than in fetal liver of NNK/EtOH-treated hamsters (Table 1Go). Although this suggests that more NNK or more NNK metabolites, including NNAL, are reaching fetal tissues in EtOH-treated hamsters, the results were not statistically significant. Both NNK and NNAL were present in fetal pancreatic tissues (Table 2Go). The levels of NNAL were 22 times higher than levels of NNK; EtOH exposure had no effect on the levels of NNK or NNAL in fetal pancreatic tissues.



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FIG. 1. Metabolic pathways of NNK metabolism.

 

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TABLE 1 NNK Metabolites Present in Tissues of Pregnant Hamsters
 

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TABLE 2 Relative Importance of Each NNK Metabolic Pathway in Pregnant Hamsters
 
Determination of CYP Expression by Western Blotting and RNase Protection
Previous studies from this laboratory have demonstrated the low but detectable expression of CYP2B and 2E family members in the fetal hamster liver by both Western and Northern blot analysis (Miller et al., 1992Go). Because the metabolite analysis described above suggested that the fetal pancreas may contain enzymes that metabolize NNK, we assayed the pancreas for the expression of various forms of CYP that were either implicated in the metabolism of tobacco-specific nitrosamines (TSNAs) or were inducible by treatment with EtOH. Fetal liver was used as a positive control to check for the specificity and sensitivity of the assays.

As shown in Figure 2Go and Table 3Go, CYP1A1, 1A2, 2E1, and members of the CYP2B family were detectable by immunoblot analysis of 25 µg of hamster fetal liver supernatants. Results from three independent experiments (shown individually in Fig. 2Go and presented as means ± the standard errors in Table 3Go) demonstrated that the levels of the fetal enzymes show a great deal of sample-to-sample variation. Treatment with NNK and/or EtOH appeared to cause a small 1.4- to 4-fold induction of the levels of the enzymes, but these were not statistically significant, most likely due to the high degree of variability between samples isolated from different hamster litters. A third band with a slightly lower molecular weight was observed only in lanes 4 (EtOH/NNK) and 7 (NNK only), while the middle band attributed to CYP2B1 was absent from lane 7. Although antibodies raised against the rat CYP2B proteins cross-react with hamster proteins, it has not as yet been established that the hamster actually contains genes that are orthologous to the rat forms of CYP2B1/2. Thus, it is still not clear which, if any, of these bands represent members of the hamster CYP2B family, or whether the rat antibodies cross-react to some other forms of CYP.



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FIG. 2. Western immunoblot analysis of fetal liver cytochrome P450 isozymes. Fetal livers from individual litters were pooled and the tissues homogenized in 5 volumes potassium phosphate buffer and centrifuged at 800 x g for 10 min at 4°C. Twenty five micrograms of fetal liver supernatant was separated by 10% SDS-PAGE and transferred electrophoretically to nitrocellulose filters. The blotted nitrocellulose filters were blocked for 2 h in buffer containing 5% fat-free dried milk and incubated with primary antibody (1:2000), followed by peroxidase conjugated secondary antibody (1:4000). After initial staining, the filters were stripped and restained with each of the indicated antibodies. The immunoreactive protein was visualized by chemiluminescence on Kodak X-Omat film, according to the manufacturer's instructions.

 

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TABLE 3 Expression of Cytochrome P-450s in Fetal
 
Unlike the liver, most of these enzymes were not detected by Western blotting in the fetal pancreas. As shown in Figure 3Go, only CYP1A1 gave a clearly detectable signal with 100 µg of fetal pancreatic supernatant. Similar to the fetal liver, there was a great deal of variability in the levels of expression of the enzyme from tissue isolated from different hamster litters, with densitometric analysis of the blots yielding corrected relative absorbance values of 1.00 ± 0.32, 1.33 ± 0.21, 1.49 ± 0.31, and 1.08 ± 0.28 in control, EtOH, NNK, and NNK/EtOH-treated hamsters, respectively (values are expressed as mean ± standard error, n = 3). CYP2E1 was barely detectable above the background, but was not quantifiable due to the low levels of expression. These results agreed well with the metabolite data, which showed low and variable levels of NNK metabolites that were not affected upon treatment with EtOH. Ribonuclease protection analysis, shown in Figure 4Go for CYP1A2 and 2E1, demonstrated that the transcript levels of the CYPs were detectable but were highly variable in the liver. None of the transcripts were detectable in the pancreas in any of the treatment groups.



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FIG. 3. Western immunoblot analysis of fetal pancreas cytochrome P450 isozymes. Fetal pancreas from individual litters were pooled and the tissues homogenized in 5 volumes potassium phosphate buffer and centrifuged at 800 x g for 10 min at 4°C. One hundred micrograms of fetal pancreatic supernatant supernatant was separated by 10% SDS-PAGE and transferred electrophoretically to nitrocellulose filters. The blotted nitrocellulose filters were blocked for 2 h in buffer containing 5% fat-free dried milk and incubated with primary antibody (1:2000), followed by peroxidase conjugated secondary antibody (1:4000). After initial staining, the filters were stripped and restained with each of the indicated antibodies. The immunoreactive protein was visualized by chemiluminescence on Kodak X-Omat film, according to the manufacturer's instructions.

 


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FIG. 4. Ribonuclease protection analysis of fetal liver RNA. Fetal livers from individual litters were pooled and total cellular RNA was isolated following homogenization in guanidine isothiocyanate solution and purification by centrifugation on a CsCl cushion. RNA probes were transcribed directly from PCR products synthesized from normal genomic hamster DNA and the gel-purified RNA transcripts were labeled with psoralen-biotin. The hybridization of the labeled RNA probe with sample RNA was carried out using the RPA IIITM kit according to the manufacturer's instructions. Thirty micrograms of total RNA and probe were coprecipitated and hybridized in 10 µl hybridization buffer at 42°C for 18–24 h, followed by digestion with RNase A/RNase T1 and the protected fragment, then precipitated and separated on a 0.75 mm thick 5% acrylamide/8M urea gel by electrophoresis at a constant 250 V in 1x TBE buffer for 1.5 h. The gel was electroblotted onto a positively charged nylon membrane. Nonisotopic detection of biotinylated RNA probe was carried out using the BrightStarTM Biodetect kitTM. The membrane was incubated with the chemiluminescent CDP-StarTM reagent for 5 min and exposed to X-ray film to visualize the image. The signal was quantitated by densitometry as described for the Western blot analysis

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Previous studies from our laboratory have shown that treatment of pregnant hamsters with NNK results in the induction of a variety of tumors in the transplacentally exposed offspring (Correa et al., 1990Go). Coadministration of EtOH and NNK to the pregnant dams shifts the primary site of tumor formation from the respiratory tract to the exocrine pancreas (Schuller et al., 1993Go) and causes an increase in the overall levels of NNK metabolism in both fetal liver and lung tissues (Jorquera et al., 1992bGo). These studies prompted us to examine the levels of CYPs in the fetal pancreas to determine their potential role in the metabolic activation of NNK to reactive metabolites in the fetal compartment, and to identify the mechanism of the EtOH-mediated alterations in the site of tumorigenesis in hamsters exposed in utero to both agents.

NNK and its primary metabolite, NNAL, were both detected at low and highly variable levels in the fetal pancreas from hamsters treated with NNK alone or cotreated with NNK/EtOH. In both cases, the ratio of NNAL to NNK was over 20, and treatment with EtOH appeared to have little effect on the apparent metabolism of NNK in the fetal compartment. It is not clear from these experiments whether the NNAL was formed in the pancreas itself as a result of metabolism, or represents a biodistribution of the metabolite from other sites, in particular the fetal liver. However, the presence of NNAL in the fetal pancreas has important implications in this model, as NNAL has been shown to induce pancreatic tumors in both adult F344 Fisher rats and after transplacental exposure in fetal hamsters (Rivenson et al., 1988Go; Schuller et al., 1993Go).

Similar results were obtained by Western and ribonuclease protection assays and are the first to demonstrate the presence of CYP in the fetal pancreas; members of the CYP1A family have been demonstrated previously in the islets of Langerhans in adult rat pancreas by immunohistochemistry (Clarke et al., 1997Go). As demonstrated by immunoblot analysis in Figures 2 and 3GoGo and Table 3Go, the levels of CYPs are low and highly variable in the developing fetus. Treatment with NNK and/or EtOH resulted in small increases in the levels of these enzymes in the fetal liver which, due to the high levels of variability, were not statistically significant. These results are similar to earlier observations in pregnant hamsters treated with EtOH measuring both catalytic activities (Jorquera et al., 1992bGo) and protein and RNA levels (Miller et al., 1992Go). In the present study, only CYP1A1 could be detected by Western analysis in the 15-day-old fetal pancreas, and there was no evidence for any effect of either EtOH and/or NNK on the levels of this isozyme.

Although these results initially suggest that metabolic activation of chemical carcinogens, in particular TSNAs, may not be a critical mechanism that determines the carcinogenicity of these compounds in the transplacentally exposed fetus, further studies are required to definitively document this. The apparent lack of effect of EtOH on the metabolism of NNK in both the fetal liver and pancreas is particularly intriguing, given the dramatic shift in the primary site of tumorigenesis in hamsters receiving cotreatment with EtOH (Schuller et al., 1993Go). Along these lines, recent studies from this laboratory have found that the transplacentally induced pancreatic tumors lack alterations in both the Ki-ras and p53 genes (Zhang et al., 1999Go) despite the fact that these are common events in both human and chemically induced adult rodent pancreatic tumors (Almoguera et al., 1988Go; Cerny et al., 1992Go; Chang et al., 1995Go; Hruban et al., 1993Go; Pellegata et al., 1994Go; Rozenblum et al., 1997Go; Sugio et al., 1996Go). Mangold et al. (1994) have shown that the prevalence of Ki-ras mutations in pancreatic tumor cell lines transformed in vitro by N-methylnitrosourea or N-(2-hydroxypropyl)nitrosourea differs depending on the dose, frequency of administration, and duration of exposure to the carcinogen. In addition, a more recent study has also found that Ki-ras mutations are not essential for in vitro carcinogenesis by BOP-treated hamster pancreatic ductal cells (Ikematsu et al., 1997Go). It has also been reported that patients who both smoked and drank had a lower risk for Ki-ras mutations in their pancreatic adenocarcinomas than those who only smoked or only drank, suggesting an interaction between alcohol and tobacco (Malats et al., 1997Go).

However, metabolism of NNK to DNA-binding electrophiles still cannot be ruled out by the data presented thus far. In particular, determination of DNA adduct levels are required to directly assess the potential for NNK-mediated damage to pancreatic DNA. Small differences in metabolism-mediated DNA adduct formation could play a role in determining the alterations in target organ specificity observed with EtOH coadministration. It is interesting that the cocarcinogenic effects of EtOH were seen mainly in the fetal pancreas and adrenal glands (Schuller et al., 1993Go). Future studies will focus on potential signal transduction pathways that may mediate epigenetic events in transplacentally induced pancreatic cancer.


    ACKNOWLEDGMENTS
 
The authors are grateful to Nathalie Harvey for excellent technical assistance in the metabolism study. This project was supported in part by grant RO1 CA42829 to HMS and Cancer Center Support Grant P30 CA12197 from the National Cancer Institute, which provided support for the Wake Forest University Comprehensive Cancer Center Analytical Imaging Core Facility, the DNA Synthesis Core Laboratory, and the DNA Sequencing and Gene Analysis Facility.


    NOTES
 
1 Present address: Department of Internal Medicine, Interfaith Medical Center, 555 Prospect Place, Brooklyn, NY 11238. Back

2 Present address: Department of Pathology, Abbott Laboratories, Abbott Park, IL 60064–6104. Back

3 To whom correspondence should be addressed at the Department of Cancer Biology, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157. Fax: (336) 716-0255. E-mail: msmiller{at}wfubmc.edu. Back

4 Although the hamster cytochrome P-450 RNAs and proteins cross-hybridize to rat DNA probes and antibodies, it has not yet been established that the hamster actually contains genes that are orthologous to the rat forms of CYP2B1/2. We have therefore referred to the hamster RNA transcripts and proteins identified by Western blot analysis as hamster CYP2B1 and 2B2 based on their mobilities in polyacrylamide gels, which were comparable to the rat 2B1 and 2B2 forms. At this time, there is no evidence to indicate that these hamster forms have the same catalytic specificity as the rat isozymes. It should also be noted that we have no evidence to indicate whether the forms found in the hamster pancreas and liver are necessarily the same isozymic forms. Back


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 MATERIALS AND METHODS
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 DISCUSSION
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