Birth defects are the leading cause of infant mortality and a major source of childhood morbidity. Birth defects encompass not only structural defects, but also functional deficits, particularly common in the central nervous system. The etiology of most birth defects remains a mystery. Certain birth defects appear to be more common within families, suggesting a genetic component. Yet, relatively few birth defects are known to be associated with a readily identifiable single gene defect. Exposure to high levels of some environmental compounds has been associated with birth defects, yet not all offspring that are exposed appear to be affected. Thus, it is likely that the combination of genetic makeup and environmental exposure determine the risk for teratogenesis. This concept of gene-environment interaction is the focus of the recently established MCW/CHW Birth Defects Research Center.
Interaction between ADH2 genotype and ethanol intake in pregnancy as risk factors An example of this gene-environment concept can be seen in my initial studies which centered on the outcome of offspring exposed antenatally to ethanol. Among the offspring of chronic alcoholic women who continue drinking during pregnancy, less than 10% will have the most severe morbidity, the fetal alcohol syndrome. The mechanism of varying susceptibility of exposed offspring may include genetic-or environmentally-induced variation in ethanol metabolizing enzymes. Ethanol is metabolized by two enzyme systems, alcohol dehydrogenase (ADH) and cytochrome P4502E1 (CYP2E1). We have previously shown that a particular allele, ADH2*3, is associated with offspring protection, measured as both better neurodevelopmental outcome and greater birth weight (McCarver et al., 1997). We chose to focus on this particular genetic difference because the enzymes determined by the alleles at the ADH2 locus vary widely in their kinetic constants. In addition, the allele with the greatest disparity (ADH2*3) is only found in the African American population which is more susceptible to alcohol related birth defects. For this study, we prospectively determined the alcohol intake in more than 10,000 African American women during pregnancy. From these pregnant women, we recruited a population of more than 1500 women for determination of ADH2 genotype using allele specific oligonucleotide probing of PCR-amplified DNA. Using a stratified design, we selected about 350 women, based on their periconceptional alcohol intake and ADH2 genotype, who were followed through pregnancy and delivery and until one year postpartum when we assessed their infants' development. Our observation of a specific genetic risk factor was the first observation of such a risk factor for alcohol related birth defects. The database and bank of DNA obtained in this study are being used currently in studies of the other enzyme that oxidizes ethanol, CYP2E1.
Genotypic and Phenotypic Variation in CYP2E1 CYP2E1 is constitutively expressed in human liver and brain, and ethanol-induced increases in CYP2E1 have been demonstrated in both of these organs. Importantly, CYP2E1 is present in the brain in areas known to be affected in fetal alcohol syndrome. Moreover, the enzyme is expressed in human fetal cephalic tissue early, by about 8 weeks post-conception. This contrasts with ADH which is only present in trace amounts in the brain. In recently completed phenotypic studies, we demonstrated that CYP2E1 metabolicability varies about 8-fold among African American women of childbearing age, and we confirmed that both ethanol and obesity are associated with increased CYP2E1 metabolic ability.
Pursuing the hypothesis that intersubject variation in ethanol-induction varies and may be a risk determinant, we identified a new genetic polymorphism in the regulatory region of CYP2E1 that is associated with increased metabolic ability in the presence of ethanol consumption or obesity (McCarver et al., 1998). Women who are drinkers and have the mutation have significantly greater CYP2E1 metabolic ability than women who are drinkers and lack the mutation. Interestingly, the 96-base pair insertion mutation is relatively common, but the incidence varies across ethnic groups, occurring in about 30% of African Americans and about 7% of Caucasians.
My currently NIH funded studies involve evaluating the mechanism for this polymorphism using in vitro DNA/protein binding assays and using reporter gene constructs to evaluate alterations in gene transcription. In addition, we are determining the impact of this CYP2E1
mutation on infant outcome following intrauterine exposure to ethanol using the database and DNA bank from our studies on ADH.
Role of CYP2E1 in risk of adverse outcome following organic solvent exposures In addition to metabolizing ethanol, CYP2E1 is responsible for the metabolism and activation of many organic solvents. Intrauterine exposure to one of these solvents, toluene, is known to cause a syndrome similar to that of fetal alcohol syndrome. We are beginning studies which will test the interaction between solvent exposure, ethanol intake during pregnancy, CYP2E1 genotype and CYP2E1 metabolic ability as risk factors for offspring neurodevelopmental outcome. We will be enrolling a large population of mother-infant pairs prospectively during pregnancy based on the mother's documented exposure to toluene and the mother's CYP2E1 genotype. The assay for toluene, using gas chromatography-mass spectroscopy with purge and trap injection, yields a sensitivity in the ppt range and also allows us to assess the mother's exposure to 30 other organic solvents. To assess alterations in maternal CYP2E1 metabolic ability during pregnancy, we will use a recently developed western blot technique to assess the concentration of the enzyme in white blood cells. Thus, this study using multiple approaches, will be the first to objectively measure important exposures during pregnancy and test potentially important interactions among multiple exposures and both genetic and phenotypic metabolic ability. In future studies, we plan to evaluate another category of organic solvents, the halogenated hydrocarbons, which are implicated in the pathogenesis of congenital heart defects.
In summary, my research interest is genetically-determined and environmentally-induced differences in xenobiotic metabolism as risk factors for birth defects. In pursuing this interest, I am using multiple approaches and tools, including epidemiologic methodology, clinical outcome assessment, molecular techniques and chemical analysis. Such diversity enhances my ability to address complex clinical questions, as well as to engage in multidisciplinary interactions with others with similar goals.
Whetstine JR, Yueh M-F, Hopp KA, McCarver DG, Williams DE, Park C-S, Kang J-H, Cha Y-N, Dolphin CT, Shephard EA, Phillips IR, and Hines RN: Ethnic differences in human flavin-containing monooxygenase 2 (FMO2) polymorphisms: Detection of expressed protein in African Americans. Toxicol. Appl. Pharmacol. 168, 216-224, 2000.
McCarver DG: ADH2 and CYP2E1 genetic polymorphisms: Risk factors for alcohol-related birth defects. Drug Metab Disp 29:562-565, 2001.
Park C-S, Kang J-H, Chung W-G, Yi H-G, Pie J-E, Park D-K, Hines RN, McCarver DG, Cha Y-N. Ethnic differences in allelic frequency for two flavin-containing monooxygenase 3 (FMO3) polymorphisms: Linkage and effects on in vivo and in vitro FMO activities. Pharmacogenetics 12, 77-80, 2002.
Hines RN and McCarver DG. The ontogeny of human drug metabolizing enzymes: Phase I oxidative enzymes. J Pharmacol Exp Ther 300, 355-360, 2002.
McCarver DG and Hines RN. The ontogeny of human drug metabolizing enzymes: Phase II conjugation enzymes and regulatory mechanisms. J Pharmacol Exp Ther 300, 361-366, 2002.
Zheng Y-M, Henne KR, Charmley P, Kim RB, McCarver DG, Cabacungan ET, Hines RN and Rettie AE. Genotyping and site-directed mutagenesis of a cytochrome P450 meander Pro-X-Arg motif critical to CYP4B1 catalysis. Toxicol. Appl. Pharmacol. 186:119-126, 2003.
Hines RN, Luo Z, Hopp KA, Cabacungan ET, Koukouritaki SB and McCarver DG. Genetic variability at the human FMO1 locus: Significance of a yin yang 1 element polymorphism (FMO1*6). J. Pharmacol. Exp. Ther. 306:1210-1218, 2003.
Johnsrud EK, Koukouritaki SB, Divakaran K, Brunengraber L, Hines RN and McCarver DG. Human hepatic CYP2E1 expression during development. J. Pharmacol. Exp. Ther. 307:402-407, 2003.
Ou J, Ou Z, McCarver DG, Hines RN, Oldham KT, Ackerman AW and Pritchard KA, Jr. Trichloroethylene decreases heat shock protein 90 interactions with endothelial nitric oxide synthase: Implications for endothelial cell proliferation. Toxicol. Sci. 73:90-97, 2003.
Koukouritaki SB, Manro JR, Marsh SA, Stevens JC, Rettie AE, McCarver DG and Hines RN. Developmental expression of human hepatic CYP2C9 and CYP2C19. J. Pharmacol. Exp. Ther.308:965-974, 2004.
Cronk CE, Malloy ME, Pelech AN, Millere RE, Meyer SA, Cowell M and McCarver DG. Completeness of state administrative databases for surveillance of congenital heart diseases. Birth Defects Res. (A) 67:597-603, 2003.
McCarver DG. Applicability of the principles of developmental pharmacology to the study of environmental toxicants. Pediatrics 113:969-972, 2004.
Cronk CE, Pelech AN, Malloy ME and McCarver DG. Excess birth prevalence of hypoplastic left heart syndrome in eastern Wisconsin for birth cohorts 1997-1999. Birth Defects Res. (A) 70:114-120, 2004.
Yauck JS, Malloy ME, Blair K, Simpson PM and McCarver DG. Proximity of residence to trichloroethylene-emitting sites and increased risk of offspring congenital heart defects among older women. Birth Defects Res. 70:808-814, 2004.
Das UG, Cronk CE, Martier SS, Simpson PM, McCarver DG. Alcohol dehydrogenase 2*3 affects alterations in offspring facial morphology associated with maternal ethanol intake in pregnancy. Alcohol Clin. Exp. Res., 28:1598-1606, 2004.