Transplacental Lung Carcinogenesis: A Pharmacogenetic Mouse Model for the Modulatory Role of Cytochrome P450 1A1 on Lung Cancer Initiation

Research in

JULY/AUGUST 1994 VOLUME 7, NUMBER 4

0 Copyright 1994 by the American Chemical Society

Invited Review

Transplacental Lung Carcinogenesis: A Pharmacogenetic Mouse Model for the Modulatory Role of Cytochrome P450

1Al on Lung Cancer Initiation Mark Steven Miller*

Molecular Carcinogenesis Laboratory, Department of Pathobtology, University of Tennessee, College of Veterinary Medicine, Knoxville, Tennessee 37901

Received February 7, 1994

Lung cancer is the leading cause of cancer-related deaths of both men and women in the United States. Lung neoplasms tend to be resistant to standard radiation and chemotherapeutic regimens, with overall survival rates of less than 15% (1). Current research has focused on the molecular pathogenesis and etiology of the disease with the goals of developing novel, mechanism-based chemotherapeutic agents targeted to specific genetic lesions and chemopreventative strategies to prevent the occurrence of the disease.

Cigarette smoke has been documented as a major etiologic agent in human lung tumorigenesis (2, 3). In addition, a recent epidemiological study has provided evidence for an association between the levels of fine- particulate air pollutants and deaths due to lung cancer (4). Thus, lung cancer can be considered an "environmental disease", the result of the inhalation of toxic chemicals found ubiquitously in the environment that subsequently cause genetic damage to lung tissue. Recent advances in molecular oncology have demonstrated the important interactions between environmental factors and host genetics, including differences in drug metabolic capacity, in determining the susceptibility of individuals to lung cancer induction (5-8). Several constituents of tobacco smoke and industrial pollution, including poly-

* Telephone: 615-974-8206, FAX Number: 615-974-5616; E-mail: MILLER.MARK @ hospital.vet.utk.edu.

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cyclic aromatic hydrocarbons BAHs),' nitrosamines, and heterocyclic amines, have been implicated as potential etiologic agents in lung cancer induction (2). This review will focus on the induction of lung cancer following in utero exposure to PAHs in pharmacogeneticmouse models as a paradigm for human lung cancer.2 Because of some of the unique properties of the fetus, transplacental carcinogenesis protocols allow the investigator to conduct research on the role of cytochrome P450 1Al (P450 1Al) in lung cancer initiation that would be difficult to do in adult animals. Several good reviews on the effects of chemical toxicants and their metabolism during the prenatal period have previously been published (9-12). Although this review will focus on the initiation phase of tumorigenesis, with particular emphasis on the metabolism- mediated formation of DNA adducts and subsequent formation of mutagenic lesions, the reader should be aware that PAHs can have other effects on the exposed organism. The reader is referred to a recent study by Puga et al. (13) elucidating the potential tumor-promoting effects of

'Abbreviations: AHH, aryl hydrocarbon hydroxylase; BNF, B-naphthoflavone; BP, benzo[alpyrene; P450, cytochrome P460; EROD, ethoxyresorufin 0-deethylase; GST, glutathione S-transferase; MC, 3-methylcholanthrene; PAH, polycyclic aromatic hydrocarbon; RFLP, restriction fragment length polymorphism; SCLC, small cell lung cancer; kb, kilobase. *As with any review article, I had to be judicious in my selection of

references. I often referenced review papers or the more recent literature as representative of a particular research area, aa it waa not possible within the scope of this article to do an exhaustive literature review. I therefore apologize to my colleaguesfor any sins of omission or commission.

1994 American Chemical Society

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compounds that bind to the Ah receptor.

Chem. Res. Toxicol., Vol. 7, No. 4, 1994 Miller

P450 1Al in Mice and Men

Many cancer-causing chemicals are highly lipophilic molecules that require metabolic conversion to more polar compounds in order to be eliminated from the body. The P450 monooxygenase system represents the initial detoxification step leading to the eventual elimination of toxic compounds. Exposure to certain substrates of P450 leads to an inductive response, whereby the organism increases the levels of specific P450 enzymes (14 , 15). PAHs are ubiquitous environmental toxicants, and exposure to these compounds results in the induction of P450 1 A l and 1A2 (16-19). The induction of these drug metabolic enzymes leads to enhanced metabolism of the toxicants and their more rapid excretion from the body. Unfortunately, these P450 enzymes are also responsible for the conversion of the parent compound to its ultimate, carcinogenic metabolites. This results in an increase in reactive inter- mediates that can cause damage to cellular macromol- ecules, such as DNA. Thus, induction of P450 1Al and 1A2 can be a double-edged sword, leading to the increased elimination of toxic chemicals on the one hand and the metabolic activation of these compounds to reactive mutagens on the other.

The induction of the P450 1A family of enzymes is mediated by the Ah receptor, which has recently been cloned and shown to be a member of the bHLH superfamily of transcription factors (20,21; reviewed in ref 22). Binding of the PAH ligand to the Ah receptor activates the receptor to a DNA binding form, and the receptor-ligand complex then translocates into the cell nucleus where it interacts with specific DNA sequences that mediate an increase in the rate of P450 1 A l transcription (16, 19, 23). Early studies on the regulation of P450 1 A l transcription by the Ah receptor were conducted in rodents. However, subsequent studies have demonstrated the high degree of similarity in the structure and regulatory mechanisms of the mouse and human P450 1 A l genes (24-30). The expression of the human P450 1 A l gene is regulated by the same mechanisms operative in mice-namely, binding of the hydrocarbon substrate to the Ah receptor, resulting in a transcriptional increase of P450 1 A l expression and subsequent increase in translated protein (15,16,19,23) .

An extensive literature exists on the consequences of P450 1Al-mediated metabolism of environmental chemicals in both rodent and human tissues (reviewed in refs 17 and 18). Metabolism of PAHs results in the formation of reactive electrophiles, which can bind to DNA and cause mutations in critical gene sequences controlling cell growth and differentiation. Metabolic studies with human tissues have shown that human enzyme preparations catalyze the formation of the same DNA-carcinogen adducts found in studies using mouse tissues as the source of enzymes (31 1. Recent studies, using highly sensitive 32P-postlabeling or immunoassay techniques, have demonstrated the presence of PAH-DNA adducts in tissues from individuals who were heavy smokers or had occupational exposure to chemical carcinogenic agents (32-35). The consequences of these increased adduct levels have been shown to result in more damage to DNA. Perera et al. (35) were able to show that increased levels of PAH-DNA adducts were associated with an enhanced rate of sister chromatid exchange, higher levels of chromosomal aberrations, and a doubling of the number of patients demonstrating

overexpression of ras protein. Vrieling et al. (36) found that increased adduct levels were correlated with an increased frequency of mutations in the hprt gene locus in lymphocytes from smokers compared to nonsmokers.

Recent advances in molecular oncology have begun to delineate the actual genes damaged as a result of exposure to chemical carcinogens. I t is becoming readily apparent that the same genes shown to be mutated in human cancers also exhibit similar mutations in mouse model systems. Mutations of the Ki-ras gene have been found in approximately 30-4094 of human non-small cell lung cancers (SCLC) (37-39). Subsequent studies with larger patient populations have shown that the majority of mutations occur in the 12th codon of Ki-ras, with G - T transversions of the first or second base of the GGT wild-type sequence predominating (40,41). Mutations at the 61st codon were not seen as frequently, but tended to exhibit CAA -. CGA transitions. These mutations in human Ki-ras genes are identical to those found in chemically-induced mouse lung tumors following exposure to PAHs such as benzo[alpyrene (BP) (42-44). Clearly, similar mechanisms of pathogenesis are operative in the two species.

Epidemiological Studies Linking P450 1Al Expression with Lung Cancer

The first studies to establish a link between expression of P450 1Al and lung cancer incidence were performed by Kellermann et al. (4.59, who demonstrated that high levels of aryl hydrocarbon hydroxylase (AHH) inducibility were found in patients with bronchogenic carcinoma. These results were confirmed with AHH assays utilizing cryopreserved lymphocytes (46). Postmitochondrial supernatants from lung cancer patients exhibited significant alterations in the levels of several enzyme activities, including AHH (47). A subsequent study showed that lung microsomes from smokers exhibited 3-fold greater levels of AHH activity than microsomes prepared from the lungs of nonsmokers, and it was found that enzyme activity in the lymphocyte preparations did not necessarily correlate with that found in lung microsomes (48). This may explain some of the discrepancies noted by several groups studying the association between enzyme activity and lung tumor susceptibility (reviewed in refs 5-81, Recent studies by McLemore et al. (49) have found elevated levels of ethoxyresorufin 0-deethylase (EROD) activity and P450 1Al RNA in normal lung tissue from smokers compared to nonsmokers. The lung tumor tissues generally exhibited abnormal regulation of P450 1 A l RNA levels. In contrast to Toussaint et al. GO), who suggested that P450 1Al expression was decreased in tumor tissue, McLemore et al. found that a significant number of primary tumor tissues had induced levels of P450 1Al RNA (49). Fifty percent of the tumors contained an unusual 10-kb P450 1Al RNA transcript, not found in normal tissue from the same individual.

Although an association between the levels of P45O 1Al- dependent metabolism and lung cancer incidence have been suggested in these studies, the molecular mechanisms regulating this effect are still not well understood. Many of these studies suffer from the typical problems encoun- tered when using human tissues, as the degree of inter- individual variability in drug metabolic activity was fairly large, making statistical comparisons difficult. Studies in humans are also complicated by the variability in genetic background of the human population. In addition to P450

Invited Reviews Chem. Res. Toxicol., Vol. 7, No. 4, 1994 473

responsive (AhbAhb), to induction of P450 1 A l by PAHs, while DBA/2 mice are noninducible, or nonresponsive (AhdAhd), due to the presence of a low-affinity binding form of the Ah receptor (71). The Ah receptor allele segregates as an autosomal dominant trait, with crosses between the two strains of mice yielding a phenotypically responsive litter consisting of the hybrid genotype (AhbAhd):

(1) C57 (AhbAhb) X DBA (AhdAhd) - F1 (AhbAhd) all responsive3

(2) DBA (AhdAhd) X C57 (AhbAhb) A further backcross between the inducible F1 generation and the noninducible DBA strain will yield a second generation litter in which half of the offspring are responsive and half are nonresponsive to P450-inducing agents:

(3) F1 (AhbAhd) X DBA (AhdAhd) - 50 7% responsive 50 7% nonresponsive

(4) DBA (AhdAhd) X F1 (AhbAhd) The flexibility of this model is readily apparent. In

crosses 1 and 3, the fetuses gestate within an inducible mother, whereas in crosses 2 and 4, the fetuses reside within a noninducible maternal environment. Through a series of reciprocal genetic crosses, it is possible to generate experimental conditions where both inducible and noninducible fetuses are gestating in either of the two maternal environments. Since fetal mice with different abilities to metabolize PAHs (either inducible or noninducible for P450 1Al) will be residing within the same maternal environment, the experimental design allows one to determine how individual genetic differences in drug metabolic capacity observed in the fetal and maternal compartments can influence the incidence and mechanism of tumor causation in the transplacentally-treated offspring. By exposing fetuses differing in their P450 1 A l content to carcinogens in utero, one can eliminate many of the variables that are introduced when separately treating animals from two totally different strains.

As is the case with human lung cancers (72)) the induction of lung tumors in mice is strongly influenced by their genetic background (73). Mouse strains can be divided into three groups on the basis of their sensitivity to both spontaneous and induced lung tumors-high, intermediate, and resistant. Both inbred C57 and DBA strains are resistant to lung tumor formation (73). Thus, similar to human lung tumorigenesis, these particular strains of mice have a low spontaneous rate of tumor formation and thus generally produce lung tumors in response to environmental chemical exposures, making this an excellent model in which to mimic the environmental and genetic factors that influence the formation of human lung tumors. The fact that both the C57 and DBA mice are resistant to the induction of lung cancer suggests that they share similar genetic backgrounds for this trait.

Several studies have indicated that a t least three separate genetic loci influence lung tumor susceptibility in different strains of mice. These loci have been termed the pulmonary adenoma susceptibility (Pus) genes (reviewed in refs 73 and 74). The Pus-1 gene, which

1A1, other genetic loci have been implicated in modulating individual susceptibility to lung tumorigenesis. In particular, specific alleles of the glutathione S-transferase (GST) Ml(51-54)) P450 2D6 (55-57)) andp53 (58) genes may all contribute to lung cancer susceptibility in the human population. Thus, while a strong correlation exists between the levels of P450 1Al RNA and metabolism of P450 1A1, as measured by the AHH and EROD assays, the degree to which P450 1Al expression correlates with lung tumor incidence in the human population is still an open question.

Researchers are beginning to elucidate, a t the molecular level, the possible mechanistic basis for the association of certain P450 1 A l phenotypes with the incidence of lung cancer. Kawajiri et al. (59) have identified a MspI restriction fragment length polymorphism (RFLP) at the 3’ end of the P450 1Al gene that appears to correlate with susceptibility to lung cancer. Subsequent studies have found that this RFLP also cosegregates with a high inducibility phenotype in cultured lymphocytes (60). However, the simultaneous examination of all three parameters-AHH activity, RFLP, and lung tumor incidence-has not been examined in the same study. A 46211e - Val mutation in the 7th exon of the P450 1 A l gene (at the heme binding site of the protein) has also been detected and appears to be closely linked with the MspI polymorphism (61). Following the identification of the MspI polymorphism, Nakachi et ul. found that individuals with the susceptible genotype exhibited an extremely high risk toward lung cancer a t low-dose levels of cigarette consumption, and this difference in risk between the different genotypes decreased with increasing cigarette dose (62)) suggesting that a t higher levels of exposure other pathways may become involved in the metabolism of aromatic hydrocarbons. In addition, it was found that the presence of the susceptible genotypes for both the P450 1 A l and GSTMl genes had a synergistic effect on the risk for lung cancer (63,64), although recent studies in other laboratories have failed to find an association between PAH-DNA adduct levels and P450 1 A l genotype (65-68).

These studies suggest a possible explanation, a t the molecular level, for the genetic basis for the association of high P450 1Al metabolic activity with lung tumor incidence. However, the multiple genetic loci responsible for determining susceptibility to lung cancer in the human population, combined with the high degrees of inter- individual variation, make it difficult to establish the relative contribution of P450 1 A l in lung cancer initiation. For these reasons, several laboratories have turned to mouse model systems to further delineate the role played by P450 1Al in lung tumorigenesis.

The Transplacental Pharmacogenetic Mouse Model-A Paradigm for the Genetic Basis of

Metabolism in Human Lung Cancer Pathogenesis

The characterization of a pharmacogenetic mouse model has allowed the examination of the role of P450 1 A l gene induction on the modulation of lung tumor susceptibility (69,70). While several strains of mice have been developed that differ in their inducibility for P450 1A1, the C57BL/6 and DBAI2 are perhaps the most widely studied pair of this group. C57BL/6 mice are genotypically inducible, or 3The maternal strain is expressed first in all genetic crosses.

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appears to be the predominant determinant of lung tumor susceptibility, has not been identified but appears to be intrinsic to the lung tissue itself, as determined by experiments with lung tissue explants (75, 76). One susceptibility locus, Pus-2, has recently been identified as the mouse Ki-rus2 gene (77). Resistant and susceptible strains of mice exhibit a RFLP at the Ki-ras2 locus, with resistant mice containing a 0.7-kb EcoRI fragment and susceptible mice a 0.55-kb gene fragment (77). Both C57 and DBA mice demonstrate identical restriction patterns a t this locus, consistent with both strains being relatively resistant to tumor induction (73). The Pus-3locusappears to be related to the major histocompatibility complex and may be a gene that resides a t this particular region of the genome (reviewed in ref 73).

It appears that differences in metabolic capacity segregate genetically from the Pus loci, allowing an examination of the role of drug metabolic phenotype separately from these other genetic factors. The results of studies with backcrossed C57 and DBA mice have shown that adult mice, in contrast to fetally exposed animals, do not exhibit differences in lung tumor incidence despite phenotypic differences in their metabolic capacity (78). Responsive adult mice exhibited only a 4- to 7-fold induction of pulmonary AHH activity following treatment with 3-methylcholanthrene (MC) whereas responsive fetal mice exhibited a 25- to 50-fold induction of metabolism in the lung. This provides further support for P450 1Al playing a key role in determining tumor susceptibility in this fetal mouse model and argues against the possible influence of other genetic factors. The fact that the Pus genes segregate from the P450 1 A l locus strongly suggests that this model should provide us with important and useful information on the role of metabolic phenotype in determining susceptibility to lung tumor induction.

The pharmacogenetic mouse model thus exhibits several traits that make it a good paradigm for human lung cancer. The mice have a low incidence of spontaneous lung tumors and, similar to humans, develop tumors in response to chemical toxicants. They have a well-characterized genetic difference in their metabolic phenotypes as a result of the inheritance of a defective Ah receptor in the DBA mice (71), which segregates in a simple Mendelian fashion. This makes experimental manipulation of the model and interpretation of the data a far simpler matter compared to epidemiological studies in the human population. As will be discussed below, treatment during the transplacental period results in a high incidence of lung tumors; thus it is possible to test agents that may inhibit or enhance the carcinogenic effects of various chemicals using moder- ate-sized experimental groups. Finally, since mice are given only one or two doses of carcinogen over a very limited time period, time course studies to assess genetic lesions associated with initiation, promotion, and tumor progres- sion can be performed without the complications introduced by a multiple dosing regimen.

The use of a protocol involving in utero exposure to carcinogenic chemicals may be quite applicable to potential human exposure routes. Epidemiological studies have suggested a link between maternal exposure to carcinogenic agents, particularly PAHs and nitrosamines, and increased cancer incidences in the offspring, but have generally ignored the possible effects of maternal smoking. In the few instances where correlations have been made between maternal smoking habits and tumor formation in the

Miller

offspring, childhood tumors such as leukemia and lym- phomas are used as the end point for tumor causation. Very few studies have examined the population at risk in middle age, where the occurrence of lung cancer would be expected on the basis of latencies observed in animal model systems. In the only such study known to this investigator (791, however, it was shown that the tumor incidence for several organ sites in adults was increased as a result of transplacental or early passive childhood exposure to a smoking environment, where one or both parents smoked. Recent studies have shown a positive correlation between maternal exposure to potentially carcinogenic agents and the incidence of childhood tumors. Transplacental exposure to either hair dyes (80) or cigarette smoke (81, 82) was associated with increased incidences of Wilms’ tumor (80, 81) and hematological cancers (81, 82) in children under 15 years of age. Another study has recently demonstrated the presence of PAH-DNA adducts in fetal tissues from mothers who were nonsmokers, suggesting that these environmental compounds are capable of crossing the placental barrier and damaging DNA in the human fetal population (83). PAH adducts to DNA (84) and hemoglobin (85) were shown to be higher in both placental and fetal tissues from smokers compared to nonsmokers. Elevated levels of BP-DNA adducts found in the placentas of pregnant women who smoked correlated with high levels of BP hydroxylase activity (86). Clearly, this area of transplacental carcinogenesis has not received adequate attention given the potential risks to the fetus of mutagenic and carcinogenic damage by environmentally prevalent toxicants.

Ontogeny of P450 1Al Expression in Mice4

The characterization of the pharmacogenetic mouse model in 1972 (69, 70) began a series of experiments, conducted in several independent laboratories, aimed at determining the role of P450 1Al in cancer initiation. Interest in the transplacental carcinogenesis of PAHs was heightened by the early studies of Nebert and Gelboin (89, who were the first to demonstrate the transplacental induction of mouse AHH activity in various organs, including the liver and lung, by MC. Subsequent studies demonstrated the induction of hepatic AHH activity as early as the 12th day of gestation following injection of either MC, BP, or 0-naphthoflavone (BNF) to pregnant mice (88-91). Using both functional enzyme assays and polyacrylamide gels to identify specific bands, Guenthner and Nebert provided evidence that the induction of hepatic P450 1Al by environmental compounds occurred earlier in gestation than did inducibility of P450 1A2 (92). These early results were confirmed at the molecular level by the use of specific cDNA probes (24) to the P450 1 A l and 1A2 genes. Nebert and his co-workers demonstrated that induction of hepatic P450 1Al was evident by day 12 of gestation, whereas induction of the P450 1A2 transcripts did not occur until parturition (93). Indeed, a subsequent

‘In the discussion that follows, the reader should be aware that not all laboratories define the first day of gestation by the same method. Some laboratories call the first day that the vaginal plug is observed day 1, while others call this day 0. Some laboratories do not look for vaginal plugs and refer to the day after presumed mating aa either day 0 or 1. In this review, I used the original terminology of the authors from the papers that I have cited, since it waa not always possible to determine what method was employed to date the pregnancy. Thus, the geetation days reported herein can vary by 1 day, depending on the definition used by the individual groups.


mother was inducible for P450 1Al. A subsequent study from the same laboratory also demonstrated the importance of the route of administration as a determinant of the incidence of PAH-mediated teratogenic and toxic events in the fetuses (101). Administration of a 120 mg/ kg dose of BP by oral gavage to pregnant, noninducible AKR mothers on days 2 and 10 of gestation resulted in a greater incidence of toxicity and teratogenicity in the noninducible offspring than in their inducible littermates. As was the case for the ip route of administration, no differences were seen when the fetuses were gestating in an inducible maternal environment (AhbAhd). The lack of a notable difference in fetal susceptibility when the mother was responsive to PAH-mediated induction of P450 1Al may be explained by the low levels of fetal P450 1Al in early gestation, as noted above (87-92,95-97). At these early time points in the prenatal period the inducibility of fetal P450 1Al is very poor. Thus, the inducible mother may metabolize enough PAH so that the responsive fetuses are not exposed to doses adequate to induce the levels of their enzyme much above that seen in their nonresponsive littermates. When the mother is noninducible, however, a much greater portion of PAH may cross the placental barrier and attain concentrations high enough to increase the levels of fetal P450 1Al.

Blake et al. (102) used in vitro bacterial mutagenicity assays to demonstrate that fetal liver homogenates could catalyze the formation of mutagenic metabolites from BP. These authors treated outbred CD1 mice and inbred mice exhibiting high ((2571, intermediate (C3H), or low (DBA) inducibility for P450 1Al with polychlorinated biphenyls on day 13 of gestation and then isolated fetal livers, whole fetuses minus the livers, maternal livers, and placentas 4 days later. The authors found that although fetal liver homogenates had only 5-10% of the mutagenic activity seen in maternal livers, there was a positive correlation between the number of revertants per milligram of fetal protein and fetal AHH activity across the 4 strains. More importantly, it was found that the mutagenic activities of the placenta and the whole fetus minus the liver were lower than that of fetal liver preparations and that their mutagenic activities did not exhibit any correlation with AHH activity, supporting earlier hypotheses that the toxic effects of PAHs on the fetus were mediated by fetal enzyme. These studies also demonstrated that induction of higher levels of P450 1Al in late gestation could result in the formation of mutagenic metabolites following exposure to PAHs.

The results of these early studies clearly pointed to fetal P450 1Al-mediated metabolism of PAHs as a modulating factor in determining host susceptibility to the toxic and teratogenic effects of these environmental chemicals. The data obtained from these short-term in vivo and in vitro toxicity studies provided the first clues suggesting that P450 1Al may playa role in determining host susceptibility to the carcinogenic effects of these toxicants as well. York and Manson (103,104) were among the first to examine the role of P450 1Al in modulating both the toxicity and carcinogenicity of transplacental exposure to PAHs. These authors performed backcross experiments in which noninducible DBA female mice were mated with inducible B6D2F1 males (AhbAhd), resulting in a litter in which the ratio of inducible to noninducible fetuses was 1:l and all fetuses resided in a nonresponsive maternal environment. The pregnant mothers were treated by oral gavage with

study (94) showed that induction of AHH and acetanilide 4-hydroxylase activity, substrates for the P450 1Al and 1A2 genes, respectively, coincided with the results obtained by polyacrylamide gels (92) and Northern blot analysis (93).

Although the inducibility of the P450 1 A l gene was not evident until the 12th day of gestation, a low level of constitutive RNA expression was observed as early as gestation day 7 in the fetal liver (95). Expression of P450 1A2 RNA, however, was not seen until gestation day 17, indicating that this P450 enzyme did not play a role in the fetal metabolism of PAHs early in gestation. Pedersen et al. (96) reported that exposure to BP resulted in increased levels of sister chromatid exchanges in tissues of both embryonic and extraembryonic origin in postimplantation- stage embryos (gestation days 7.5-8.5). However, a subsequent study, utilizing in situ hybridization techniques, found that P450 1 A l was not expressed in embryonic tissue until 10.5 days of gestation (97), suggesting that extraembryonic sites of carcinogen metabolism were responsible for these genetic lesions in early gestation. Inducible expression of P450 1Al was seen by gestation day 12 in the lung and liver, but was not observed in any other extrahepatic organs. These studies suggest that, during early gestation, metabolism of foreign chemicals is mediated exclusively by extraembryonic tissues and reflects the metabolic phenotype of the mother rather than that of the individual fetus. However, by late gestation (i.e., after gestation day 15) the levels of fetal P450 1 A l are high enough to contribute significantly to the metabolism of PAHs.

Transplacental Lung Carcinogenesis Following characterization of the pharmacogenetic

mouse model and the discovery of the simple Mendelian inheritance of the responsive phenotype, several laboratories initiated experiments to determine the role of P450 1 A l in modulating the carcinogenicity, teratogenicity, and toxicity of foreign chemicals. Kouri et al. (981, using adult mice, first demonstrated in 1974 that inducible mice exhibited a 12-fold higher incidence of subcutaneous skin sarcomas following sc injection of MC than did their noninducible littermates. In one of the first transplacental exposure experiments to be performed, Lambert and Nebert (99) demonstrated that asingle ip injection of either MC or dimethylbenz[alanthracene between days 5 and 13 of gestation resulted in a somewhat greater incidence of congenital defects (i.e., fetal resorptions, stillbirths, clubfoot, crown to rump length) in inducible compared to noninducible offspring. A more comprehensive study utilizing the inducible C57 and noninducible AKR mouse strains demonstrated that treatment with BP doses ranging from 50 to 300 mg/kg on days 7 and 10 of gestation caused significant differences in fetal toxicity and teratogenesis that correlated with fetal P450 1 A l inducibility (100). In experiments utilizing reciprocal backcrossed mice, the inducible (AhbAhd) offspring showed increased incidences of stillbirths, fetal resorptions, congenital abnormalities, and low birth weights compared to their noninducible littermates when the mother was nonresponsive. These toxic and teratogenic lesions were associated with higher levels of BP binding to DNA in the inducible fetuses of noninducible mothers. However, these differences in fetal toxicity and teratogenicity following BP administration were not observed when the pregnant


various doses of MC, ranging from 7 to 63 mg/kg/day, on gestation days 15,16, and 17. The authors found a dose- dependent increase in the toxic effects of MC in both inducible and noninducible offspring following in utero exposure to the carcinogen. The inducible littermates had higher incidences of neonatal toxicity within the same exposure group than did their noninducible counterparts (103). When the carcinogenic effects of MC exposure were examined 6 months later, the lung was found to be the major site of proliferative lesions. Interestingly, it was found that the noninducible offspring had significantly higher incidences of pulmonary nodules, adenomas, and diffuse bronchial hyperplasia5 than did the responsive offspring (104).

A similar tumor study utilizing reciprocally backcrossed mice and a single high ip injection of MC on day 17 of gestation clearly demonstrated the influence of both the maternal and fetal phenotypes on the formation of lung adenocarcinomas and adenomas in the transplacentally- treated offspring, although this study found that lung tumor incidence correlated positively with the responsive phenotype (105). In this study, a 1:l ratio of responsive and nonresponsive mice was treated in both inducible and noninducible maternal environments with several different doses of MC ranging from 5 to 100 mg/kg. Similar to York etal. (103,104), theseauthorsalsofoundadose-dependent increase in lung, as well as liver, tumor incidence with increasing MC doses (105). Within the same treatment group, the responsive offspring of both sexes were more susceptible to lung tumor initiation than their nonresponsive littermates regardless of the phenotype of the mother. In addition, liver tumors in male responsive mice were also significantly higher than in noninducible male mice. The offspring of the nonresponsive DBA mothers tended to have higher tumor incidences than the progeny of responsive B6D2F1 mothers, suggesting that, because of the lower metabolic capacity of the DBA mothers, their fetal compartments are exposed to higher levels of the carcinogen. Indeed, whereas the pregnant C57 mothers can tolerate a dose of 100 mg/kg MC, doses above 30 mg/ kg in the DBA mothers resulted in a high abortion rate. Thus, the most susceptible animals were the responsive fetuses of the nonresponsive mothers. It appears that once MC enters the fetal compartment, the higher levels of P450 1A1 induced in responsive fetuses results in agreater conversion of MC to carcinogenic metabolites that can bind DNA, resulting in a higher tumor incidence in the responsive offspring. However, inducible mothers were able to eliminate or detoxify more of the carcinogen from their body before it was able to enter the fetal compartment, accounting for the lower tumor incidence in offspring of responsive mothers. Indeed, pretreatment of different strains of pregnant mice with PNF on day 15 of gestation reduced the lung tumor incidence following MC treatment on day 17 of gestation in the offspring from inducible mothers as a result of the induction of P450 1Al and the enhanced metabolism of PAHs (106, 107). Whether the differences in the correlation of lung tumor incidence with either the responsive (105) or nonresponsive (104) phenotype of the mice is a result of the route of administration, as suggested by Legraverend et al. (101), remains to be determined. Further analyses a t the biochemical and molecular levels may elucidate the mechanisms responsible for this difference.

Miller

A subsequent study by George and Manson (108) examined the metabolism and accumulation of 14C-labeled MC by the C57 and DBA mice pretreated on day 15 of gestation with PNF. The initial levels of MC-associated radioactivity were identical in the two strains of pregnant mice, suggesting that the absorption of MC from the gastrointestinal tract was similar. As expected, the adult C57 mice cleared the MC more rapidly than their noninducible DBA counterparts. In the C57 fetal mice, the MC reached peak concentrations 2 h after administration in both the liver and lung and then started to decline. In DBA fetuses, MC continued to accumulate in the fetal lung out to 8 h after administration.

Although these results may provide an explanation for the higher susceptibility of fetuses from noninducible mothers for lung neoplasias, the use of different strains of inbred mice that were not genetically crossbred makes it difficult to assess what implications this may have for the situation where fetuses differing in their inducibility for P450 1 A l are residing within the same maternal environment. The use of PNF complicates their interpretation since pretreatment with inducers of P450 1Al was not a feature of the tumorigenicity bioassays. Future studies employing backcrossed mice will be essential in order to determine the pharmacokinetic and biochemical basis for the observed differences in fetal susceptibility to lung tumor induction.

Studies at both the biochemical and molecular levels have shown that fetal mice display remarkably different induction properties than adult mice. Miller et al. (78, 109,110) employed the inducible B6D2F1 fetuses (AhbAhd) resulting from a parental cross (C57BL/6 X DBAI2) to examine the effect of in utero MC exposure on AHH activity and P450 1Al and 1A2 induction. BP hydroxylase activity was used as a marker for P450 1Al expression. Since BP is metabolized by several forms of P450 (Ill), the contribution of other forms of P450 in mediating BP metabolism could be assessed.

Treatment with MC resulted in a dose-dependent, maximal induction of AHH activity by 8 h after injection in both the fetal liver and lung (1091, in contrast to the longer 16- to 24-h time frame observed in adult liver preparations (112). The enzyme data correlated well with RNA blot analysis, as MC caused maximal induction of P450 1 A l RNAlevels 4 h after injection in both fetal organs. Interestingly, MC appeared to cause a biphasic induction of both AHH activity and P450 1 A l expression in the lungs. In addition, the induction of P450 1A2 RNA was demonstrated in fetal liver and, for the first time, in fetal lung, although at very low levels relative to P450 1Al. The results indicated that the increase in functional AHH activity was primarily due to induction of P450 1Al . Differences in the observed induction kinetics suggest that P450 1Al exhibits tissue- and age-dependent specificity, with induction occurring much earlier in fetal than adult liver preparations. These age-related differences in fetal inducibility have also been found in fetal rats (113).

The maternal phenotype appears to play an important role in influencing the induction kinetics of both AHH activity and P450 1Al RNA levels (110). As expected, although basal AHH activity was comparable in maternal C57 and DBA livers, maternal DBA liver activity was not increased after treatment with inducing agents. In experiments utilizing reciprocal crosses between the two parental strains, the absolute levels of AHH activity and


the relative induction ratios in fetal lungs from DBA mothers (D2B6F1) were very similar to those obtained in fetal tissues from C57 mothers (B6D2F1 fetuses) 8 and 24 h after injection of a 30 mg/kg dose of MC. This implied that, in both DBA and C57 mothers, enough MC and PNF reached the fetus to cause a comparable induction of AHH activity. However, by 48 h after injection of MC, the levels of pulmonary AHH activity of B6D2F1 fetuses had declined to nearly control values, whereas in D2B6F1 fetuses, AHH activity remained elevated over the activity found in control tissue samples. Analysis of RNAs from both B6D2F1 and D2B6F1 tissue samples confirmed the enzymology data. The D2B6F1 fetuses generally showed a more sustained inductive effect over a longer time period than the B6D2F1 fetuses. The results also confirmed the biphasic nature of the inductive effect in fetal lung tissues discussed earlier, again demonstrating the tissue-specific effects discussed above.

It thus appears that the metabolic and pharmacokinetic parameters of both mother and fetus combine to strongly influence the susceptibility of the fetus to chemically- induced tumors. The genetic responsiveness of the mother and fetus to induction of drug metabolic enzymes by PAHs plays an important role in determining the relative levels of activated vs detoxified metabolic products that cross into the fetal compartment. Differences in pharmacokinetic distribution and clearance of various substrates from the maternal and fetal compartments also appear to influence susceptibility to tumor initiation by chemical carcinogens. It is highly likely that the lower levels of maternal metabolism in DBA mice, due to their inability to induce drug metabolic enzymes in response to exposure to environmental toxicants, allow more parent compound to reach the fetus over an extended time period, allowing a sustained exposure to MC in D2B6F1 fetuses with the resulting increases in tumorigenicity noted in earlier tumor studies (105).

Several studies have shown that fetal tissues are more sensitive to the toxic and carcinogenic effects of chemical and physical carcinogens than are adult tissues (78; reviewed in ref 114). Adult mice must be treated with at least three 100 mg/kg doses of MC in order to cause lung tumors, while treatment of a pregnant mother with only a single dose causes tumor formation in the offspring at lower concentrations (78,105). In contrast to the transplacental lung tumor model, studies in backcrossed adult mice have shown no differences in the incidence of lung tumors in responsive and nonresponsive mice (78). The difference in the correlation of lung tumor incidence to the inducibility phenotype of the individual in fetal vs adult tissue may be partially explained by their very different biochemical properties.

With the exception of MC-inducedlung tissue, the fetus generally exhibits much lower levels of AHH activity than the adult (78). Constitutive adult lung AHH activities in 8OOg supernatants were 4- to 27-fold greater than constitutive fetal enzymatic activities. In the adult, liver 8OOg supernatants displayed enzyme activities that were 12- to 200-fold higher than comparably treated fetal liver supernatants.

When treated with MC, adult and fetal enzymatic turnover numbers in lung were practically identical 48 h after injection [6.00 f 2.70 vs 6.52 i 0.91 pmol of 3- OH-BP formed/(min.mg of protein) in fetal and adult tissue, respective1y)l , as confirmed by immunoblotting,

and there was less than a 2-fold difference 8 and 24 h after injection, indicating that adult lung tissue also demon- strates near-maximal induction of P450 1 Al-mediated AHH activity by 8 h after MC administration (78). This suggested that, in the fetal lung, gene regulatory mechanisms are already in place that allow full induction of P450 1Al to adult levels. The 4- to 27-fold increases in adult constitutive pulmonary AHH activity are probably due to the increased expression of other P450 enzymes. Thus, a t 24- and 48-h postinjection, MC causes a 25-fold increase in the levels of AHH activity in the fetal lung. In the adult lung, the higher constitutive level of AHH activity was maximally increased only 7-fold by MC treatment while the absolute induced activity remains constant relative to fetal values. Clearly, the decreased relative inducibility of adult lung tissue is due to the increased expression of another enzyme@) that metabolizes BP but is resistant to induction by MC. This form@) of P450 may play a role in the detoxification of BP, resulting in the greater resistance of the adult to lung tumor initiation by PAHs. These observations clearly suggest that metabolic phenotype plays a key role in determining susceptibility to PAH-induced lung tumorigenesis. Thus, the results obtained in the transplacental mouse model correlate well with epidemiological studies in humans implicating P450 1Al as a modulator of lung tumor susceptibility in different individuals.

Consequences of P450 1Al-Mediated Metabolism: Future Trends

The convergence of data from rodent carcinogenicity and human epidemiological studies clearly point to P450 1 A l as an important determinant of individual risk to lung tumor susceptibility following exposure to environmental chemicals. Current and future studies have begun to examine the molecular mechanisms of lung tumor pathogenesis in both humans and experimental animal models, although a paucity of data currently exists regarding tumor induction during the fetal period.

Bolognesi et al. (115) have shown that in utero exposure to BP results in the induction of DNA damage in CD1 mouse fetal lung and liver tissues, as measured by the alkaline elution technique, and the amount of genetic damage increased when the pregnant mothers were pretreated with Aroclor 1254, a potent inducer of mouse P450 1Al . These results correlate well with the in vivo carcinogenicity assays and biochemical analyses of P450 1 A l induction and lung tumor incidence. The presence of adducts to several PAHs has been demonstrated by the 32P-postlabeling technique in fetal ICR mice (116). The adduct levels in lung tissue increased with gestational age, suggesting that adduct levels may correlate with the relative levels and inducibility of P450 1Al (117). In addition, the fetuses of mice exposed to both sidestream and mainstream cigarette smoke exhibited increased levels of sister chromatid exchanges and micronuclei formation (118).

The activation of protooncogenes and inactivation of tumor suppressor genes are commonly observed events in both human and experimentally induced rodent lung tumors (42, 44). Recent studies have demonstrated the occurrence of mutations at the Ki-ras gene locus in approximately 30% of non-SCLCs (37-39), while mutations at the p53 gene locus are one of the most frequently found mutations in all human cancers, including lung (1 19,


120). While mutation of these critical cell regulatory molecules has been implicated in the pathogenesis of human and rodent lung tumors, little work has been done with mouse models following in utero exposure to environmental toxicants. A recent study by Loktionov et al. (121) has demonstrated the presence of mutations in the Ki-ras gene in 40% of mouse lung tumors induced by treatment with dimethylbenz [a] anthracene. Considering the marked sensitivity of the fetus to tumor induction by chemical carcinogens and the long latency for actual tumor formation, fetal exposures to chemical carcinogens may account for a significant portion of tumors that occur later in adult life. Since fetal exposure protocols require only a single injection of carcinogen in order to cause significant tumor yields, transplacental rodent models will allow the investigator to employ serial sacrifice protocols to delineate when during neoplastic transformation certain types of genetic damage occur. In addition, use of the pharmacogenetic mouse model described in this review will allow a correlation of P450 1Al expression with both qualitative and quantitative alterations in the mutational spectrum produced by PAHs. Recent studies have shown that the types of mutations observed in oncogenic loci can vary in a dose-dependent manner (122-125). This may provide a molecular basis for the differences in tumor susceptibility observed in inducible and noninducible mice. The recent development of strains of mice congenic for the Ah receptor locus (1 26) may prove to be a particularly valuable research model for these types of studies.

Miller

Conclusion

Epidemiologic studies have provided evidence for the association of elevated P450 1 A l levels with lung tumor incidence and have suggested a link between maternal exposure to carcinogenic chemicals and early childhood cancers. I t is clear that both environmental and genetic factors interact to modulate the individual’s response to environmental carcinogens. However, given the complex genetics of the human population and the myriad of potential toxicants each individual may be exposed to, it becomes difficult to define the importance of any one genetic factor against the background of so many variables. The pharmacogenetic mouse model offers an unique opportunity to delineate the role of P450 1 A l in lung tumorigenesis. Similar to humans, the mice have a low incidence of spontaneous lung tumors and develop tumors in response to chemical toxicants. The genetic basis of their different metabolic abilities has been well characterized. Treatment during the transplacental period results in a high incidence of lung tumors, making this model particularly well suited to test for possible chemopreventative agents or novel antineoplastics. In addition, administration of carcinogens during the perinatal period allows the investigation of the pathogenesis of the disease, as single treatment time points may be utilized. Previous results obtained from transplacental studies have demonstrated the important role played by P450 1Al in modulating lung, as well as liver, tumor incidence in mice differing in their inducibility to PAHs. The use of this model should provide valuable information on the pathogenesis of lung tumors and the modulatory role of P450 1 A l in lung tumorigenesis. This model will continue to serve as an important in vivo system to test for potential fetal carcinogens.

Acknowledgment. The author would like to thank Dr. Hildegard Schiiller for many helpful discussions and words of encouragement, and Dr. Michael McEntee for critically reviewing the manuscript.

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Transplacental Lung Carcinogenesis: A Pharmacogenetic Mouse Model for the Modulatory Role of Cytochrome P450 1A1 on Lung Cancer Initiation