Grantee Research Project Results
Final Report: Activation of Ki-ras During Transplacental Carcinogenesis
EPA Grant Number: R829428Title: Activation of Ki-ras During Transplacental Carcinogenesis
Investigators: Miller, Mark Steven , Cline, J. Mark , Manderville, Richard A. , Ross, Jeffrey A. , Townsend, Alan J. , Kock, Nancy D.
Institution: Wake Forest University School of Medicine , U. S. Environmental Protection Agency
EPA Project Officer: Aja, Hayley
Project Period: October 1, 2001 through September 30, 2004 (Extended to September 30, 2005)
Project Amount: $902,111
RFA: Children's Vulnerability to Toxic Substances in the Environment (2001) RFA Text | Recipients Lists
Research Category: Children's Health , Human Health
Objective:
Organ- and strain-specific differences in the levels of toxicant metabolism and/or DNA repair may determine the relative susceptibility of the developing organism to genetic damage that leads to the initiation of cancer. Several studies have shown that the developing organism is very sensitive to chemical and physical carcinogens, suggesting that exposure of pregnant women to environmental toxicants may place the embryo and fetus at higher risk for the induction of cancer. Despite this higher sensitivity and increased vulnerability, few studies have examined the mechanisms of cancer causation and toxic responses to environmental chemicals during gestation. The main goal of this research project was to elucidate the biochemical and molecular mechanisms that determine oncogenic damage and modulate susceptibility to chemical carcinogens during the sensitive period of fetal development.
Summary/Accomplishments (Outputs/Outcomes):
We previously demonstrated that offspring from either a D2 x B6D2F1 backcross (Wessner, et al., 1996; Leone-Kabler, et al., 1999) or from parental Balb/c mice (Gressani, et al., 1999) had high incidences of lung tumors 6 to13 months after transplacental exposure to 3-methylcholanthrene (MC). Balb/c mice exhibited a reduced latency for lung tumor formation, as the mice exhibited a 100 percent tumor incidence 6 months after a 45 mg/kg dose of MC compared to an 84 percent tumor incidence at 12 to 13 months in inducible [D2 x B6D2F1]F2 backcrossed mice treated with 30 mg/kg of MC. The two strains also exhibited a markedly different mutational spectrum in the Ki-ras gene. Whereas G→T transversions were found in 84 percent of lung lesions from [D2 x B6D2F1]F2 backcrossed mice with mutations, 62 percent of the Ki-ras mutations in lesions from Balb/c mice were G→C transversions. Although the mechanism mediating this difference in the ras mutational spectrum is unknown, possibilities include differences in the metabolism of MC resulting in either increased activation of MC to reactive electrophiles as a result of induction of Cyp1 genes or decreased detoxification due to differences in Phase II enzymes, and/or differences in adduct formation and/or repair of adducts. Alternatively, it is possible that these strain-specific effe5cts may be the result of contributions from other genetic mechanisms. Thus, we compared the effects of in utero treatment with MC on lung tumor induction in the offspring of intermediately susceptible Balb/c (Bc), resistant C57BL/6 (B6), and reciprocal crosses between these strains.
Pregnant mice were treated with 45 mg/kg of MC on day 17 of gestation. Tumor incidence and multiplicity were determined 12 to18 months after birth. Tumor incidences in Bc mice and reciprocal crosses between the two strains were 86 percent and 100 percent, respectively, while B6 mice demonstrated a remarkable resistance to tumorigenesis (Table 1), with a tumor incidence of 11 percent (p < 0.0001 for B6 vs. the other three strains). Bc, B6Bc, and BcB6 mice exhibited significant tumor involvement in the lungs; in many cases, multiple tumors coalesced into single large masses with the majority of these lesions classified as adenocarcinomas (ACs). Counting only lesions that were discrete, individual nodules, tumor multiplicities in Bc, B6Bc, BcB6, and B6 mice were 3.3 ± 3.2, 5.8 ± 3.2, 5.0 ± 2.7, and < 0.1, respectively. Pair-wise strain comparisons in the study group revealed that the tumor multiplicity in the B6 strain was significantly lower than the other three strains (p < 0.0005). All other pair-wise comparisons between strains were significant (p < 0.02) with the exception of BcB6 vs. B6Bc, which was not significant (p = 0.40). B6Bc and BcB6 mice tended to exhibit a higher incidence of spontaneous lung tumor formation, as the reciprocally crossed mice had tumor incidences in the olive oil treated controls of 13 to 22 percent compared to that seen in B6 (0%) or Bc (3%) mice, although these differences were not significant.
Table 1. Lung Tumor Formation in Crosses Between B6 and Balb Mice Treated in Utero With MC
Tumor Incidence | Tumor Multiplicity | Size of Tumors >1 mm | |
B6 x B6 | 5/46 (11%) | < 0.1 | 1.3 " 1.1 |
Balb x B6 | 25/25 (100%) | 5.0 " 2.7 | 1.5 " 0.8 |
B6 x Balb | 19/19 (100%) | 5.8 " 3.2 | 1.8 " 0.9 |
Balb x Balb | 30/35 (86%) | 3.3 " 3.2 | 1.4 " 0.8 |
On day 17 of gestation, mice were treated with a single i.p. injection 45 mg/kg of MC dissolved in olive oil. At various times after birth ranging from 12 to 18 months, the mice were killed by CO2 asphyxiation/exsanguination and lung tumors were fixed in 10% formalin and embedded in paraffin. Tumor multiplicity and size is reported as the mean " standard deviation.
Ki-ras mutations occurred chiefly in the Ks allele (96%) and were found in 79 to 81 percent of B6Bc and BcB6 mice, 64 percent of Bc mice, and 50 percent of B6 mice, with Val12, Asp12, and Arg13 mutations associated with more aggressive tumors, as demonstrated in two previous studies by our laboratory. Across all four strains in this study, G→C transversions accounted for the smallest portion of mutations (25%), which is similar to results obtained previously in [D2xB6D2F1]F2 mice whereas the incidence of G→T transversions were similar to our earlier study with Bc mice, comprising 45 percent of mutations observed. The remaining 30 percent of mutations were G→A transitions, a mutation seen previously at much lower rates (0-8%) in our earlier studies. Some of the differences between the current and previous studies may be due to the fact that the tumors in this study were isolated at later time points. Thus, a larger percentage of the tumors were adenomas (ADs) or ACs, and our previous studies have shown that hyperplastic lesions were more likely to contain the G→C mutation associated with the substitution of Cys for Gly at codon 12.
Although it is not surprising that there are differences in lung tumor susceptibility between the B6 and Balb mice, given the documented genetic differences between the two strains at the Pas1 and Papg1 loci, we were quite surprised to find such a large difference between the tumor incidences obtained previously with the [D2 x B6D2F1]F2 backcrossed mice and the parental B6 mice. We also examined tumor incidence and multiplicity in parental D2 mice and found that, despite their poor inducibility for Cyp and inability to upregulate the metabolic activation of MC, this strain exhibited a higher lung tumor incidence (25% with an N = 8) than the parental B6 strain. The fact that B6 mice had such a low tumor incidence (11%, Table 1), whereas backcrossed [D2 x B6D2F1]F2 mice with the inducible Ah phenotype exhibited a tumor incidence greater than 80 percent, clearly suggests the presence of a polymorphic gene locus between the B6 and the D2 mice that is a major determinant of tumor susceptibility following in utero exposure to environmental chemicals. Although interpretation of the crosses between the B6 and Balb mice is complicated by the documented differences at the Ki-ras and Papg1 loci, it is clear that B6 mice are highly resistant to the induction of lung tumors following in utero exposure to MC and that this resistance can be overridden by the presence of an unknown modifier gene in the D2 genome.
We examined induction of Cyp1a1 and Cyp1b1 in fetal lung and liver tissue by quantitative fluorescent real time PCR. The levels and induction of Cyp1a1, while differing somewhat between the four strains, did not appear to account for the marked differences in tumor phenotype (Xu, et al., 2005). We observed that Cyp1b1 RNA was not induced in the parental B6 lung at any time point, but was maximally induced by approximately 10-fold in the other three strains. However, this is probably not the cause of the differences in susceptibility to transplacentally induced tumors because: (1) Cyp1a1 is present in much higher concentrations than Cyp1b1 and should thus be the enzyme primarily responsible for MC metabolism; (2) other laboratories have suggested that human CYP1B1 does not mediate the activation of the 11,12-diol of MC in a mutagenesis assay; and (3) adduct levels formed following MC injection are similar in all four strains, as discussed below. We also measured the ability of maternal liver to metabolize MC to determine if subtle differences in maternal metabolism could influence the amount of carcinogen available that could cross into the fetal compartment. We measured five specific metabolic products of MC, including the 1-OH, 1-one, 2-OH, 11,12-diol, and 11,12-dione and found that, although there were some differences in the amount of specific products formed across each of the four strains, none of these differences in the maternal metabolism of MC could account for the marked differences in tumor induction.
We screened for GST enzyme activity and for expression of the individual GST α, π, μ, and θ isoforms in murine fetal lung and liver tissues. Using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate, we found that treatment with MC had no effect on the levels of GST enzyme activity in either the fetal lung or liver in any of the four strains of mice examined. Low levels of expression of each of the four isozymes were detected by Western blotting in both fetal lung and livers in all four strains. A statistically significant 3.5-fold induction was observed only for GSTμ in the fetal lung of the parental strain of Bc mice 48 hours after exposure to MC. None of the other isozymes showed any significant differences in the levels of expression following exposure to MC. Although strain-specific differences in the expression of the GST isozymes that were independent of MC treatment were observed, they could not account for the differences previously observed in either the Ki-ras mutational spectrum or lung tumor incidence in the different strains of mice. The results are consistent with previous studies (reviewed in Miller, et al., 2004) showing low levels and poor inducibility of phase II enzymes during gestation, and demonstrate for the first time that all four of the major GST isozymes are expressed in fetal tissues.
We also measured the levels of MC adducts and their disappearance from lung tissue on gestation days 18 and 19 and postnatal days 1, 4, 11, and 18 (Xu, et al., 2005). Surprisingly, it was the parental B6 mice which showed the highest levels of adducts 2 (gestation day 19) and 4 (postnatal day 1) days after injection, although this was not statistically significant. In general, there were no statistically significant differences across the strains as determined for each of the three individual spots detected with a few exceptions: (1) when total adduct levels for all three spots were combined, the levels of adducts in Balb/c x B6 mice (BcB6) were significantly lower than the levels found in the other three strains 21 (postnatal day 18) days after injection; and (2) when the spots were broken down individually, spots 2 and 3 were significantly lower in BcB6 mice than in B6Bc and parental Balb/c mice 21 days after injection; parental B6 mice did not differ statistically from any of the other three strains when the three spots were considered individually. These results confirm the data obtained on the levels of CYP and GST in the four strains, and clearly demonstrate that differences in metabolic activation or detoxification of MC do not lead to differences in MC adduct formation that could account for the differences in lung tumor susceptibility observed in parental B6 mice relative to the parental Bc or B6Bc and BcB6 F1 hybrids.
We also used a subset of mice to demonstrate the utility of using a modern clinical computer tomography (CT) system for the visualization and measurement of lung tumors in vivo. CT correctly identified the presence or absence of tumors in 6 of the 8 mice analyzed and only failed, in the absence of intravenous contrast, to identify small (< 2 mm) tumors that were located very close to the heart. Following necropsy, we compared the sizes of the tumors measured with electronic calipers to the diameters obtained by the CT software and demonstrated an excellent correlation between the two methods. Our results have implications for the clinical applications of CT to smokers, as the identification of small lesions at a very early stage through routine imaging techniques could be used to enhance early detection.
These studies highlight the role of gene-environmental interactions in determining individual susceptibility to chemical carcinogens following exposure in utero. We anticipated that our studies would demonstrate that a combination of alterations in the activation of MC by Phase I enzymes, detoxification of reactive MC metabolites by Phase II enzymes, and/or repair of DNA adducts by DNA repair enzymes would be important determinants of the amount and types of damage at the Ki-ras gene locus. This, however, has not proved to be the case. The resistance of the C57BL/6 mice to transplacentally induced lung tumors cannot be explained by differences in induction of phase I or phase II enzymes, nor by differences in adduct levels and repair. Put in context with our previous studies demonstrating that [D2 x B6D2F1]F2 inducible mice have a tumor incidence of greater than 80 percent, our results provide evidence for the existence of a novel, dominantly acting susceptibility/resistance locus that specifically confers susceptibility to chemical carcinogens during the sensitive fetal period.
References:
Wessner LL, Fan M, Schaeffer DO, McEntee MF, Miller MS. Mouse lung tumors exhibit specific Ki-ras mutations following transplacental exposure to 3-methylcholanthrene. Carcinogenesis 1996;17(7):1519-1526.
Leone-Kabler S, Wessner LL, McEntee MF, D’Agostino RB, Jr, Miller MS. Ki-ras mutations are an early event and correlate with tumor stage in transplacentally-induced murine lung tumors. Carcinogenesis 1997;18(6):1163-1168.
Gressani KM, Leone-Kabler S, O’Sullivan MG, Case LD, Malkinson AM, Miller MS. Strain-dependent lung tumor formation in mice transplacentally exposed to 3-methylcholanthrene and post-natally exposed to butylated hydroxytoluene. Carcinogenesis 1999;20(11):2159-2165.
Journal Articles on this Report : 4 Displayed | Download in RIS Format
Other project views: | All 13 publications | 4 publications in selected types | All 4 journal articles |
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Type | Citation | ||
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Miller MS. Transplacental lung carcinogenesis: molecular mechanisms and pathogenesis. Toxicology and Applied Pharmacology 2004;198(2):95-110. |
R829428 (Final) |
not available |
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Xu M, Miller MS. Determination of murine fetal Cyp1a1and 1b1 expression by real-time fluorescence reverse transcription-polymerase chain reaction. Toxicology and Applied Pharmacology 2004;201(3):295-302. |
R829428 (Final) |
not available |
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Xu M, Nelson GB, Moore JE, McCoy TP, Dai J, Manderville RA, Ross JA, Miller MS. Induction of Cyp1a1 and Cyp1b1 and formation of DNA adducts in C57BL/6, Balb/c, and F1 mice following in utero exposure to 3-methylcholanthrene. Toxicology and Applied Pharmacology 2005;209(1):28-38. |
R829428 (Final) |
not available |
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Yu Z, Loehr CV, Fischer KA, Louderback MA, Krueger SK, Dashwood RH, Kerkvliet NI, Pereira CB, Jennings-Gee JE, Dance ST, Miller MS, Bailey GS, Williams DE. In utero exposure of mice to dibenzo[a,l]pyrene produces lymphoma in the offspring: role of the aryl hydrocarbon receptor. Cancer Research 2006;66(2):755-762. |
R829428 (Final) |
Exit Exit |
Supplemental Keywords:
carcinogen, fetus, vulnerability, susceptibility, metabolism, genetic predisposition, health effects,, RFA, Health, Scientific Discipline, Toxicology, Genetics, Health Risk Assessment, Susceptibility/Sensitive Population/Genetic Susceptibility, Children's Health, Molecular Biology/Genetics, genetic susceptability, cancer risk, Ki-ras , health effects, sensitive populations, carcinogenesis, childhood cancer, lead, genetic predisposition, exposure, fetus, children, susceptibility, carcinogens, children's vulnerablity, cancer risks, carcinogen, transplacental carcinogenesis, susceptability, human susceptibility, Ki-ras, pregnancy, oncogenes, environmental hazard exposures, maternal exposureProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.