Prenatal Toxicity and Lack of Carcinogenicity of 2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) following Transplacental Exposure

Kiersten M. Gressani*,{dagger},1, Sandra Leone-Kabler{ddagger}, M. Gerard O'Sullivan§, Alan J. Townsend{ddagger} and Mark Steven Miller*,{dagger},2

* Departments of Physiology and Pharmacology, {dagger} Cancer Biology, and {ddagger} Biochemistry, Comprehensive Cancer Center, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157; and § Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Minnesota, St. Paul, Minnesota 55108

Received December 9, 1999; accepted February 4, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Accumulating evidence from human and experimental animal studies indicates that consumption of heterocyclic amines (HA), derived from cooked meat and fish, may be associated with an increased incidence of cancer. Experiments were initiated to assess the role of one of these compounds, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), as a potential transplacental carcinogen, as well as to evaluate whether in utero exposure to IQ results in the induction of fetal cytochrome P4501A1 (Cyp1a1), P4501B1 (Cyp1b1), and/or glutathione S-transferase (GST). Inducible, or responsive, backcrossed fetuses resulting from a cross between congenic C57BL/6 (AhdAhd) nonresponsive female mice and C57BL/6 (AhbAhb) responsive male mice were transplacentally exposed to olive oil or 6.25, 12.5, or 25 mg/kg of IQ on day 17 of gestation. No macroscopically or microscopically visible liver, lung, or colon tumors were found in the transplacentally treated offspring by one year after birth. Ethoxyresorufin O-deethylase (EROD) and 1-chloro-2,4-dinitrobenzene assays were performed to evaluate whether transplacental exposure to IQ results in the induction of fetal Cyp1a1 and GST, respectively, in lung and liver tissues. Results showed levels of EROD and GST activity in tissues of IQ-treated mice to be very close, if not identical, to those of mice treated with olive oil. Similarly, ribonuclease protection assay data showed that the levels of Cyp1a1 and Cyp1b1 RNA in tissues of IQ-treated mice were not significantly different from those of oil-treated controls. Previous studies have shown that the developing organism expresses very low levels of Cyp1a2. Thus, in utero exposure to IQ does not lead to induction of Cyp1a1, Cyp1a2, or Cyp1b1 in the fetal compartment, thereby maintaining the low levels of these activating enzymes in the developing organism. Taken together, these data imply that, at least under the conditions employed for these experiments, IQ may not play an important role in transplacentally induced tumorigenesis.

Key Words: Cyp1a1, Cyp1b1; glutathione S-transferase (GST); heterocyclic amines; IQ; transplacental.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
The preparation of meat and fish by conventional cooking techniques has been shown to result in the formation of highly carcinogenic heterocyclic amines (HA*) (Layton et al., 1995Go; Sugimura, 1997Go; Wakabayashi et al., 1992Go). Charred parts of broiled fish and cooked meat containing HAs have been found to be potent mutagens in bacterial assays (Nagao et al., 1977Go; Sugimura et al., 1977Go), as well as strong carcinogens in laboratory animals (Schut and Snyderwine, 1999Go; Wakabayashi et al., 1992Go). In particular, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), a prototypical HA, has been found to induce liver tumors in nonhuman primate experimental models (Adamson et al., 1990Go) and at multiple sites in rodents (Ohgaki et al., 1984Go; Takayama et al., 1984Go; Tanaka et al., 1985Go).

Despite the well-documented role these dietary carcinogens play in the tumorigenic process in adults, very few studies have addressed the effects of these compounds on the unborn fetus. Brittebo et al. (1994) demonstrated that unmetabolized 2amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) could be found in fetuses and newborns following maternal exposure to this HA. In similar studies by Ghoshal and Snyderwine (1993), Davis et al. (1994), and Mauthe et al. (1998), PhIP metabolites and PhIP-DNA adducts were observed in pups exposed to lactating rats treated with this dietary compound. The importance of studying the effects of transplacental exposure to dietary carcinogens is further supported by reports, by several groups, of a direct link between maternal exposure to dietary and environmental carcinogens and an increased incidence of childhood, as well as adult, cancer (Greenberg and Shuster, 1985Go; Turusov and Tomatis, 1997Go; Zahm and Devesa, 1995Go). Moreover, an increasing amount of data suggests that neonates may be especially susceptible to the carcinogenic effects of these compounds. In a study by Dooley et al. (1992), hepatic adenomas were induced in newborn mice following an ip injection of a heterocyclic amine, PhIP, at a dose that was 5,000–10,000 times lower than that found to be effective in adults. In addition, Paulsen et al. (1999) have recently demonstrated that neonatal exposure to PhIP results in an increased incidence of intestinal tumors in Min mice. Further, several studies have demonstrated the high sensitivity of the developing organism to the carcinogenic effects of dietary and environmental toxicants, and have provided evidence that transplacental exposure to these compounds may play an important role in the initiation of tumors during fetal development (Anderson et al., 1985Go; Miller et al., 1990bGo, 1994; Rice, 1979Go; Wessner et al., 1996Go).

It is well established that transplacental exposure to such environmental toxicants as polycyclic aromatic hydrocarbons (PAHs) results in the induction of cytochrome P4501A1 (CYP1A1), and that this is mediated by the aryl hydrocarbon (Ah) receptor (Miller, 1994Go). It is not certain, however, what role this receptor and the CYP1A1 isoform might play in transplacentally induced HA carcinogenesis. CYP1A1 is an inducible cytochrome P450 enzyme expressed primarily in extrahepatic tissues (Sesardic et al., 1990Go), and is important at an early stage of development (Kimura et al., 1987Go). Expression of the 1a1 gene has been detected as early as 10 days of gestational age in the mouse (Tuteja et al., 1985Go), and intraperitoneal 3-methylcholanthrene (MC) treatment of pregnant mice has been shown to lead to transplacental induction of Cyp1a1 enzyme activity (Nebert and Gelboin, 1969Go). Cyp1a2, on the other hand, is expressed constitutively in the liver (Kimura et al., 1986Go) and its induction during development is minimally detectable until the neonatal period (Dey et al., 1989Go; Miller et al., 1989Go,1996).

Several studies have demonstrated that HAs are metabolized primarily by CYP1A2 (Boobis et al., 1994Go); however, few studies have looked at the CYP enzymes responsible for extrahepatic metabolism of HAs early in gestation. This appears important to study, considering that IQ has been shown to induce tumors in organs other than the liver, such as the lung and colon (Ohgaki et al., 1984Go; Takayama et al., 1984Go; Tanaka et al., 1985Go). Accordingly, CYP1A1, the isoform expressed in extrahepatic tissues, may play a role in mediating the metabolic activation of IQ. Studies have shown that CYP1A1 is involved in HA bioactivation (Adachi et al., 1991Go; Boobis et al., 1991Go; Crofts et al., 1998Go), but its exact involvement in this process remains unclear since others have reported conflicting results (Snyderwine and Battula, 1989Go). In addition to CYP1A1, it is possible that other forms of the CYP1 family, such as CYP1B1, may be transplacentally induced following IQ exposure. CYP1B1 has recently been shown to be expressed in fetal tissues (Hakkola et al., 1997Go), and to be involved in the activation of a number of environmental compounds including HAs (Shimada et al., 1996Go).

A multitude of studies in the literature have investigated the carcinogenic effects of HAs in the adult model. However, despite the fact that ingestion of cooked meats during pregnancy may result in a significant level of exposure in the fetal compartment, very few studies have assessed the role of these compounds in transplacentally induced neoplasia. A congenic C57BL/6 mouse strain (Lin et al., 1991Go) was utilized in the present study to determine whether in utero exposure to a prototypical HA, IQ, results in the formation of tumors in treated offspring. Further, enzymatic and biochemical analyses were performed to evaluate whether transplacental exposure to HAs results in the induction of fetal Cyp1a1, Cyp1b1, and/or GST, and what role these metabolic enzymes might play in the tumorigenic process. In order to specifically assess the effects of fetal Cyp1a1 and 1b1 activity on the toxicity and carcinogenicity of IQ, a genetic cross was utilized that resulted in inducible fetuses residing in a noninducible mother in order to minimize the contributions of maternal metabolism.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Chemicals.
IQ (98% purity) was purchased from Toronto Research Chemicals, Inc. (Toronto, Canada); olive oil, glucose-6-phosphate, glucose-6-phosphate dehydrogenase, dicumarol, NADPH, ethoxyresorufin, resorufin, and guanidine isothiocyanate were from Sigma Chemical Co. (St. Louis, MO); [{alpha}-P32]UTP (~800Ci/mmol) was from DuPont/NEN (Boston, MA); bicinchoninic acid protein assay reagent from Pierce Chemical (Rockford, IL); RPA III Kit and mouse glyceraldehyde-3-phosphate dehydrogenase from Ambion (Austin, TX); and the BioRad Protein Assay Kit II from BioRad Laboratories (Melville, NY).

Animals and treatment protocol.
A congenic C57BL/6 (B6) mouse strain was utilized in the present study to assess the transplacental carcinogenicity of IQ (Lin et al., 1991Go). C57BL/6 (AhdAhd or B6d,d) nonresponsive female mice were mated with C57BL/6 (AhbAhb or B6b,b) responsive male mice. On day 17 of gestation (day 1 being the day the vaginal plug was first detected), a single ip injection of either olive oil or 6.25, 12.5, or 25 mg/kg of IQ suspended in olive oil was administered. Three days after transplacental treatment with IQ, offspring were born and foster nursed by untreated mothers to avoid further carcinogen exposure through the mother's milk. The mice were housed for 13 months, with no further treatment, in a pathogen-free environment in plastic cages with hardwood shavings for bedding. The mice were allowed free access to water and food (AIN-76 Purified Rodent Diet, Dyets Inc., Bethlehem, PA) and a 12-h fluorescent light/dark cycle was maintained. At 13 months of age, mice were euthanized by cervical dislocation and all major organs except the brain (including the intestines) were examined grossly for lesions. Tissues were fixed in 10% formalin, embedded in paraffin, and 6 µ sections were cut with a microtome and stained with hematoxylin and eosin. Several sections of livers and lungs from all animals in the high-dose group were examined histologically, in addition to sections of the small and large intestine from selected animals, by a board-certified veterinary pathologist, using standard histopathologic criteria (Faccini et al., 1990Go). Animal studies were approved by the Wake Forest University's Institutional Animal Care and Use Committee and followed all NIH guidelines for the care, use, and euthanasia of laboratory animals.

Preparation of 800 g and S9 supernatants.
Pregnant B6d,d nonresponsive mice were treated on day 17 of gestation with 25 mg/kg of IQ or olive oil and were euthanized by cervical dislocation 8, 24, and 48 h after injection. The fetuses were removed from the mother, decapitated, and placed on ice. Fetal livers and lungs were removed, pooled, and homogenized in a 20% wt/vol 0.1 M solution of potassium phosphate (pH 7.25) for 15 s using a Polytron homogenizer. Supernatant fractions were subjected to 3 cycles of freezing in an ethanol (EtOH)/dry ice bath and thawing in a room temp water bath. Supernatants were then isolated by centrifugation at 800 g for 10 min at 4°C in a Sorvall RT6000 refrigerated centrifuge. Samples were stored at –80°C until assayed for P450 catalytic activity. A 1/10 dilution of the supernatant was assayed for protein content by the method described by Bradford (1976).

Adult mice were treated with 25 mg/kg of IQ or olive oil and were euthanized by cervical dislocation 48 h after injection. Livers were removed and homogenized in 0.25 M potassium phosphate pH 7.25/0.15 M KCl for 30 s using a Polytron homogenizer at medium speed. Supernatant fractions were isolated by centrifugation at 9000 g for 20 min at 4°C in a Sorvall RC-58 refrigerated centrifuge and stored at –80°C until use. A 1/50 dilution of the supernatant was prepared for determination of protein concentration.

Determination of ethoxyresorufin O-deethylase (EROD) activity.
Supernatant fractions prepared from fetal liver and lungs were assayed for EROD activity by a modification of the assays described by Pohl and Fouts (1980) and Lubert et al. (1985). Specifically, 100–800 µg of supernatant protein were added to a reaction mixture consisting of 1.0 M Tris–HCl, 0.5 M MgCl2, 50 mM glucose-6-phosphate, 50 U/ml glucose-6-phosphate dehydrogenase, 2 mM dicumarol, and 10 mM NADPH. The reactions were started by the addition of ethoxyresorufin to a final concentration of 5 µM in a final volume of 2 ml and incubated for 15 min at 37°C in a shaking water bath. The reactions were stopped by the addition of methanol. Following the assay, precipitated protein was centrifuged for 5 min at 4°C at 2000 RPM in a RT6000 Sorvall centrifuge. The formation of resorufin, as measured by fluorescence, was determined using a SLM-AMINCO Luminescence Spectrometer (excitation wavelength, 522 nm; emission wavelength, 586 nm).

Determination of glutathione S-transferase (GST) activity.
Isolated fetal liver and lung tissues were sonicated briefly. An aliquot was removed, resonicated for an additional 15 s, and centrifuged at 12,000 RPM for 10 min at 4°C. Supernates were assayed for GST activity at 25°C by a standard spectrophotometric assay using 1 mM 1-chloro-2,4-dinitrobenzene (CDNB) and 1 mM GSH as substrates (Habig and Jakoby, 1981Go), and activities are expressed as nmol of substrate consumed/min/mg protein (mU/mg). Protein levels were measured with bicinchoninic acid protein assay reagent (Pierce Chemical, Rockford, IL).

Determination of GST protein expression levels.
Primary antibody to GST{alpha} was prepared as described previously (Fields et al., 1998Go). Cytosolic protein samples were boiled for 5 min prior to loading. Samples were electrophoresed on a 12% SDS–PAGE gel and transferred by semi-dry electroblotting onto a nitrocellulose membrane. The blot was blocked with 5% nonfat dry milk in PBS, then probed with a 1:1000 dilution of affinity purified rabbit polyclonal anti-human GST{alpha}. The probed blot was washed 4 times in PBS, then incubated with a 1:3000 dilution of goat anti-rabbit HRP-conjugated IgG (Cappel/ICN, Costa Mesa, CA). The blot was washed again in PBS and developed using the Renaissance Chemiluminescence Kit (NEN Boston, MA).

Determination of Cyp1a1 and Cyp1b1 RNA expression levels.
Pregnant B6d,d nonresponsive mice were treated on day 17 of gestation with 25 mg/kg of IQ or olive oil and were euthanized by cervical dislocation 1, 2, 4, 8, 12, 16, 24, and 48 h after injection. The fetuses were removed from the mother, decapitated, and placed on ice. Fetal tissue RNA was purified using the standard guanidine isothiocyanate/CsCl density centrifugation method (MacDonald et al., 1987Go) as described previously for fetal tissues (Miller et al., 1989Go,1990a,b).

Ribonuclease protection assays (RPA) were performed to determine expression levels of Cyp1a1 and Cyp1b1 in RNA isolated from fetal liver and lung tissue samples. Mouse P4501A1 and P4501B1 plasmids were kindly provided by Drs Daniel Nebert (Kimura et al., 1984Go) and Colin Jefcoate (Savas et al., 1994Go), respectively. In order to synthesize a probe for the RPA assays, a 1 µl aliquot of plasmid DNA was subjected to the polymerase chain reaction (PCR) using the Perkin-Elmer GeneAmp PCR Reagent Kit. All reactions were carried out in 100 µl and consisted of reaction buffer (10 mM Tris–HCl, pH 8.0/2.5 mM MgCl2/50 mM KCl), 200 µM dNTPs (dATP, dCTP, dGTP, dTTP), and 2 units of AmpliTaq Gold (Perkin Elmer). Primers for Cyp1a1 and Cyp1b1 (DNA Synthesis Core Laboratory, Comprehensive Cancer Center of Wake Forest University) were added at a final concentration of 0.2 µM for each primer. The first 27 nucleotides of antisense primers were specific for the SP6 promoter sequence (MAXIscript In vitro Transcription Kits, Ambion, Austin, TX). The primer sequences for Cyp1a1 were 5`-TGGGCCTCAGAGAACTCCTG-3`(sense) and 5`-GGATCCATTTAGGTGACACTATAGAAGGCAGTGTCATAAACCATTTG-3`(antisense). The primer sequences for Cyp1b1 were 5`-ATGCACAACTATCTAAGAAAG-3`(sense) and 5`-GGATCCATTTAGGTGACACTATAGAAGGAAGCATTTTTCCAAGCAAG-3`(antisense). The samples were overlaid with 100 µl of mineral oil to prevent evaporation and cross-contamination of the samples. The PCR cycle parameters consisted of an initial 2 min denaturation step at 94°C, followed by 40 cycles of 1 min at 94°C, 2 min at either 55°C (for Cyp1a1) or 51°C (for Cyp1b1), and 2 min at 72°C, with a final extension step of 72°C for 7 min.

[{alpha}-32P]UTP RNA labeled probes were synthesized using an Ambion MAXIscript In Vitro Transcription Kit (Austin, TX) according to manufacturer's instructions. Briefly, reactions were carried out in a 20 µl solution of 10x transcription buffer (includes 100 mM DTT), 10 mM NTPs (ATP, CTP, GTP), 800 Ci/mmol [{alpha}-32P]UTP, 5 U/µl RNase inhibitor, 1 µg Cyp1a1 or Cyp1b1 template DNA, and 5 U/µl SP6 RNA polymerase. This reaction mixture was incubated at 37°C for 90 min. Two U/µl RNase-free DNase was added and the reaction was incubated at 37°C for 15 min to remove the template DNA. The 234 bp labeled probes were gel purified on a 5% acrylamide/8 M urea gel to separate out full-length transcripts from any prematurely terminated transcription products and unincorporated nucleotides.

Sample RNA and gel purified [{alpha}-32P]UTP labeled RNA probe were hybridized using an Ambion RPA III Kit (Austin, TX) according to manufacturer's instructions. Briefly, 5 µg of sample RNA and 1.0 x 104 cpm of labeled probe were mixed together and concentrated by ethanol precipitation at –20°C for 15 min, followed by centrifugation at 4°C for 15 min. The EtOH was carefully removed and the samples were air dried for 5 min at room temp. The pellets were resuspended in Hybridization III Buffer, heated at 95°C for 4 min, and incubated at 50°C in a dry bath overnight to permit hybridization of the probe and complementary mRNA in the sample RNA. After hybridization, RNase III Digestion Buffer/RNase T1 Mix was added and the samples were incubated for 30 min at 37°C to degrade single-stranded, unhybridized probe. RNase Inactivation/Precipitation III Solution was then added to the mixture and samples were placed at –20°C for at least 15 min, followed by centrifugation at 4°C for 15 min. Samples were then incubated at 95°C for 4 min, separated on a 5% acrylamide/8 M urea gel at 26 W for 1 h at room temperature, and visualized by autoradiography on a Molecular Dynamics Phosphorimager 445SI (Sunnyvale, CA). Mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal control and MC-treated tissue served as experimental positive controls. Expression of Cyp1a1 and/or Cyp1b1 mRNAs was quantified using ImageQuant Software (Molecular Dynamics).


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
In the present study, the potential toxicity and carcinogenicity of IQ was assessed following in utero exposure to this HA. In addition, enzymatic and biochemical assays were performed to determine whether in utero exposure to IQ leads to induction of fetal Cyp1a1, Cyp1b1, and/or GST. EROD and CDNB assays were employed to measure the levels of EROD and GST activity, respectively. RPA analyses were carried out to quantitate the levels of Cyp1a1 and Cyp1b1 RNA expression in tissues of oil- and IQ-treated mice.

An initial 6-week toxicity study was undertaken to determine the appropriate doses of IQ to be administered to pregnant mice. Doses of IQ greater than 25 mg/kg were shown to be highly toxic to the fetuses, resulting in a low rate of live births. Only 1 of the 6 litters born to mothers treated with a 50 mg/kg dose of IQ survived beyond 2 weeks. Three of the litters (50%) had no live births (all the pups born dead) while the neonates from 2 of the 3 remaining litters exhibited a "fail to thrive" syndrome, as evidenced by poor weight gain and a sickly appearance. These neonates were either euthanized or died prior to 2 weeks after birth. In contrast, 5 of 7 litters from the oil-treated and 5 of 6 litters from the 25 mg/kg dose group of IQ survived until the end of the 6-week study period. The 50 mg/kg dose group had only 12 live births out of a total of 26 pups born to 6 mothers, compared to 40 live births out of a total of 40 pups born to 6 mothers treated with 25 mg/kg of IQ, and 33 live births out of a total of 37 pups born to 7 mothers treated with olive oil. By the end of the 6-week study period, 31 of the 37 pups (84%) born to the oil-treated controls and 32 of the 40 pups (80%) born to the 25 mg/kg dose group were still alive. The body weights for the oil-treated and 25 mg/kg IQ dose groups were comparable and are shown in Table 1Go. Thus, the doses selected for the long-term tumorigenicity study were a high dose of 25 mg/kg, a medium dose of 12.5 mg/kg, and a low dose of 6.25 mg/kg. Pregnant mice were treated with the appropriate doses of IQ and the resultant carcinogenic effects in the offspring were determined.


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TABLE 1 Litter Weights of Mice Treated with Olive Oil or IQ
 
Gestational IQ exposure had no effect on maternal weight gain, length of pregnancy, birth weight, fetal mortality or postnatal weight gain (Table 2Go). Since previous studies performed in this laboratory with other carcinogens have demonstrated that fetal tissues are more susceptible to the toxic and carcinogenic effects of environmental chemicals, it was anticipated that IQ would be carcinogenic following in utero exposure. However, despite the well-documented role that IQ plays in experimental adult neoplasia (Eisenbrand and Tang, 1993Go), no tumors were observed in any organ from control or IQ-treated mice. Despite careful examination of several sections of tissue from predilection sites such as lung, liver, or intestines, the only lesions observed were some small foci of lymphocytes and neutrophils in liver, perivascular and/or peribronchial lymphocyte aggregates (sometimes prominent) in lung, and sometimes prominent gut-associated lymphoid tissue in intestine. These lesions were interpreted as incidental findings. To further test the transplacental carcinogenicity of IQ, a group of BALB/c mice, which are more susceptible to lung tumor formation than the C57BL/6 strain (Malkinson, 1989Go), were exposed in utero to either olive oil or 25 mg/kg of IQ. Following a 6-month period, the mice were euthanized by CO2 asphyxiation and checked for the presence of any tumors. Previous studies from our laboratory have shown that BALB/c mice demonstrate lung tumors 6 months after in utero exposure to MC, compared to 12–13 months for the more resistant C57BL/6 strain (Gressani et al., 1999Go; Wessner et al., 1996Go). Consistent with the results seen in the C57BL/6 mice, BALB/c mice did not exhibit any macroscopically visible lung or liver tumors, thus strengthening our observation of the inability of IQ to induce neoplasia following transplacental exposure.


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TABLE 2 Litter Numbers, Survival, and Final Weights of Oil- and IQ-Treated Mice
 
Supernatant fractions from lungs and livers pooled from transplacentally treated offspring were prepared and assayed for P450 catalytic activity. Since the expression of most forms of CYP are either very low or entirely absent during gestation, the role of CYP1A1 in the metabolism of IQ could be directly assessed in the relative absence of background levels of other forms, including CYP1A2 (Dey et al., 1989Go; Miller et al., 1989Go; Raucy and Carpenter, 1993Go). These studies were designed to specifically determine if Cyp1a1 and Cyp1b1 could mediate the first step in the metabolic activation of IQ in the absence of Cyp1a2. The induction of fetal Cyp1a1 following transplacental exposure to PAHs has been demonstrated previously, as well as the important role this enzyme plays in the metabolism of these compounds (Anderson et al., 1985Go; Miller et al., 1990bGo; Wessner et al., 1996Go). However, results from the present study indicate that fetal Cyp1a1 does not play a major role in the metabolism of IQ and that levels of 1a1 or 1b1 are not induced following in utero exposure to IQ. Levels of EROD activity 8, 24, and 48 h after injection, in both fetal lung and liver supernatants from 4 separate litters, were undetectable in tissues from both oil-and IQ-treated mice.

To ensure that the EROD assays were performed properly and that these results were accurate, adult and fetal mice were exposed to MC, a well-documented inducer of Cyp1a1 (Conney, 1982Go). Supernatant fractions from these mice were assayed using the same reagents and under the same conditions used for the IQ-treated mice. In contrast to the results obtained with IQ, 8 h after treatment with MC induction of EROD activity was observed in both lungs and livers of transplacentally treated mice, reaching levels of 2.0 ± 0.82 and 26.6 ± 6.2 pmol of resorufin formed/min/mg of protein, respectively. In addition, treatment with MC resulted in a 74-fold increase in adult EROD activity over oil-treated controls (375 ± 151.7 vs. 5.1 ± 2.01 pmol of resorufin formed/min/mg of protein, respectively). These results are in excellent agreement with our previous studies utilizing the benzo[a]pyrene hydroxylase assay (Miller et al., 1989Go,1990b), and suggest that transplacental exposure to IQ does not lead to a substantial induction of fetal Cyp1a1.

Fetal lung and liver tissues were also isolated for the determination of alterations in the levels of expression of Cyp1a1 and Cyp1b1 RNA. To test for the integrity of isolated RNA, all samples were separated and visualized on an agarose gel. Consistent with the results obtained from the EROD analyses, data from ribonuclease protection assays showed that the levels of Cyp1a1 RNA in liver (Figure I) and lung (data not shown) tissue from IQ-treated mice were identical to those found in olive oil-treated controls, using RNAs from two individual litters for each time point. Mouse glyceraldehyde-3-phosphate (GAPDH) was utilized in each experiment as an internal control to correct for any differences in the amounts of RNA added to each reaction sample. Four h MC-treated tissue served as a positive control since previous studies demonstrated maximal increases in Cyp1a1 RNA expression following a 4 h transplacental exposure period to MC (Miller et al., 1989Go,1990a,b). Accordingly, both the MC-treated liver and lung tissues exhibited, on average, a 20-fold increase in expression of Cyp1a1 RNA over oil- and IQ-treated samples (Fig. 1Go).



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FIG. 1. Liver RNA expression levels of fetal Cyp1a1 and Cyp1b1. Pregnant B6d,d nonresponsive mice were treated ip on day 17 of gestation with 25 mg/kg of IQ or olive oil and were euthanized by cervical dislocation 1, 2, 4, 8, 12, 16, 24, and 48 h after injection. Total RNA was isolated from fetal livers and ribonuclease protection assays were employed to determine RNA expression levels of Cyp1a1 and Cyp1b1. Samples were separated on a 5% acrylamide/8 M urea gel, visualized by autoradiography, and quantified on a Molecular Dynamics phosphorimager. Mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal control and MC-treated tissue served as the experimental positive control. The numbers at the top indicate times after injection of the indicated chemicals. Results are representative of at least 2 separate experiments.

 
Since no induction of Cyp1a1 was detected in lung and liver samples and the expression of Cyp1b1 was recently found to be present in fetal tissues (Hakkola et al., 1997Go), we next assayed for alterations in the levels of expression of this enzyme. Results from the RPA analyses of Cyp1b1 expression are shown in Figures 1 and 2GoGo. Similar to the Cyp1a1 data, levels of Cyp1b1 expression in lung (data not shown) and liver tissues of IQ-treated mice were found to be similar to those found in oil-treated controls. These analyses demonstrated no significant increase in Cyp1b1 RNA levels in 4 h MC-treated tissue, contrary to the 20-fold increase in Cyp1a1 RNA levels observed in these same samples (Figure 1Go). The adult MC-treated tissue did, however, exhibit induction of Cyp1b1, but at lower levels than Cyp1a1 (10-fold difference) (Fig. 2Go).



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FIG. 2. Expression levels of Cyp1a1 and Cyp1b1 in adult liver tissue. Responsive adult mice were treated with 30 mg/kg of MC or olive oil and were euthanized by cervical dislocation 12 h after injection. Total RNA was isolated from the liver and ribonuclease protection assays were employed to determine the levels of Cyp1a1 and Cyp1b1 RNA expression. Samples were separated on a 5% acrylamide/8 M urea gel, visualized by autoradiography, and quantified on a Molecular Dynamics phosphorimager. Mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal control. The numbers at the top indicate times after injection of the indicated chemicals. Results are representative of at least 2 separate experiments.

 
While it is not clear why exposure to IQ did not result in the formation of tumors, it is probable that this lack of tumor initiation was related to the low, uninducible levels of fetal metabolic enzymes. Thus, despite the fact that IQ has been shown to cause tumors in organs other than the liver (Ohgaki et al., 1984Go; Takayama et al., 1984Go, Tanaka et al., 1985Go), and that Cyp1a1 has been found to be induced by HAs (Adachi et al., 1991Go; Kleman et al., 1992Go), results obtained from the EROD and RPA analyses did not indicate a significant induction of Cyp1a1 in tissues excised from mice exposed in utero to IQ. This may account for the lack of tumorigenicity of IQ in this model. Although the levels of expression of N-acetyltransferases (NAT) are relatively low during gestation, which may also partially account for this lack of tumorigenicity, the formation of HA-adducts in rat fetuses has been demonstrated (Hasegawa et al., 1995Go), which suggests that the enzymes mediating metabolic activation of HAs are present, albeit at low levels, in the fetus.

The enzymatic activity of glutathione S-transferase (GST) was measured and utilized as a marker of electrophilic response element (ERE)-mediated induction. Fetal levels of GST activity in lung and liver tissue were assessed using 1-chloro-2,4-dinitrobenzene (CDNB). As shown in Figure 3Go, activity levels of GST in IQ-treated samples were similar to those found in olive oil-treated tissues. The average activity in oil-treated fetal lung was 57.0 ± 1.7 nmol of substrate consumed/min/mg protein (mU/mg), compared to an activity of 62.5 ± 1.1 mU/mg found in IQ-treated lung. Similarly, the average GST activity in fetal liver tissue treated with olive oil and IQ was 407 ± 36 and 410 ± 17 mU/mg, respectively. Western blot analysis additionally revealed that expression of GST{alpha} in fetal lung and liver tissue samples treated with IQ were the same as found in oil-treated tissues.



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FIG. 3. Activity levels of glutathione S-transferase (GST). Fetal liver and lung tissues were pooled and sonicated briefly. An aliquot was removed, resonicated for an additional 15 s, and centrifuged at 12,000 RPM for 10 min at 4°C. Supernates were assayed for GST activity with 1-chloro-2,4-dinitrobenzene (CDNB) at 25°C. GST activity is expressed as nmol of substrate consumed/min/mg protein (mU/mg) and represents the mean ± SD (3 litters/treatment group).

 
The data presented in this study suggest that transplacental exposure to IQ may not pose a serious cancer risk to the gestating organism in the presence of the normally low levels of drug metabolism observed in the unchallenged fetus, at least in the experimental design used in this study. Moreover, the results confirm that IQ is a poor inducer of Phase I and II enzymes and that, in the absence of CYP1A2, cannot enhance the levels of extrahepatic CYP1A1 and 1B1 enzymes to increase the production of potentially toxic metabolites. However, these studies are somewhat limited in that only one route of administration was tested, only one HA, IQ, was examined, and mice were euthanized at 13 months rather than being carried out for two years. Thus, additional studies are needed to test other HAs with different routes of administration and dosing schedules before any definitive conclusions can be drawn regarding the potential in utero carcinogenicity of these compounds.


    ACKNOWLEDGMENTS
 
The authors would like to thank Hermina Tulli for the processing of tissue samples. We also thank Dr. Colin Jefcoate of the University of Wisconsin for providing us with the plasmid for mouse Cyp1b1. This project was supported in part by grant R01 ES08252 (to MSM) from the National Institute of Environmental Health Sciences, and Cancer Center Support Grant P30 CA12197 from the National Cancer Institute, which provided support for the Analytical Imaging Core Facility and the DNA Synthesis Core Laboratory.


    NOTES
 
1 Present address: Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, NC 27157. Back

2 To whom correspondence should be addressed. Fax: (336) 716–0255; E-mail: msmiller{at}wfubmc.edu. Back


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