Grantee Research Project Results
2004 Progress Report: Activation of Ki-ras During Transplacental Carcinogenesis
EPA Grant Number: R829428Title: Activation of Ki-ras During Transplacental Carcinogenesis
Investigators: Miller, Mark Steven , Ross, Jeffrey A. , Manderville, Richard A. , Townsend, Alan J. , Cline, J. Mark
Current 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
Current 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 Period Covered by this Report: October 1, 2003 through September 30, 2004
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 objective of this research project is to elucidate the biochemical and molecular mechanisms that determine oncogenic damage and modulate susceptibility to chemical carcinogens during the sensitive period of fetal development.
Progress Summary:
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 development of cancer because of their increased vulnerability. Our research has shown that treatment of pregnant mice with 3-methylcholanthrene (MC) results in the formation of lung and liver tumors in the offspring 1 year after birth. We identified both strain- and organ-specific differences in the Ki-ras mutational spectrum in lung and liver tumors induced by in utero exposure to MC. We propose, therefore, to elucidate the biochemical and molecular mechanisms that determine oncogenic damage and modulate susceptibility to chemical carcinogens during the sensitive period of fetal development.
During the past project year, we compared the effects of in utero treatment with MC on lung tumor formation in the offspring of intermediately susceptible Balb/c (Bc), resistant C57BL/6 (B6), and reciprocal crosses between the two strains. Pregnant mice were treated with 45 mg/kg of MC on day 17 of gestation and tumor incidence, multiplicity, and tumor size determined in the offspring 14-18 months after birth. Bc, B6Bc, and BcB6 mice exhibited a 100 percent tumor incidence, whereas the resistant B6 mice had an incidence of 11 percent. B6 mice exhibited 4 small nodules 18 months after birth, whereas Bc mice rarely survived beyond 14 months and BcB6 and B6Bc mice survived to approximately 16 months. We have examined induction of Cyp1a1 and Cyp1b1 in fetal lung and liver tissue by quantitative fluorescent real time PCR, and the levels of GSTα, GSTμ, GSTθ, and GSTπ by Western blotting using isozyme specific antibodies. The levels and induction of Cyp1a1, while differing somewhat between the four strains, did not appear to account for the marked differences in tumor phenotyope. We did observe that Cyp1b1 RNA was not induced in the parental B6 lung at any time, but was maximally induced by ~10-fold in the other three strains. This is probably not the cause, however, of the differences in susceptibility to transplacentally induced tumors because (1) Cyp1a1 is present in much higher concentrations than Cyp1b1 and thus, should 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. 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. Surprisingly, it was the parental B6 mice that 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 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 x B6 (BcB6) mice 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 mice 21 days after injection; parental B6 mice did not differ statistically from any of the other three strains when the 3 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 Balb or B6Bc and BcB6 F1 hybrids. We currently are completing studies on potential differences in MC metabolism and mutations in Ki-ras. These studies highlight the important interactions between genetic and environmental factors in determining individual susceptibility to environmental toxicants during development.
Future Activities:
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 will be important determinants of the amount and types of damage at the Ki-ras gene locus. This has so far, however, proven not 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 >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.
Journal Articles:
No journal articles submitted with this report: View all 13 publications for this projectSupplemental 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, genetic susceptability, Molecular Biology/Genetics, 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.