Determination of Human Hepatic CYP2C8 and CYP1A2 Age-Dependent Expression to Support Human Health Risk Assessment for Early Ages.

Predicting age-specific metabolism is important for evaluating age-related drug and chemical sensitivity. Multiple cytochrome P450s and carboxylesterase enzymes are responsible for human pyrethroid metabolism. Complete ontogeny data for each enzyme are needed to support in vitro to in vivo extrapolation (IVIVE). This study was designed to determine age-dependent human hepatic CYP2C8 expression, for which only limited ontogeny data are available, and to further define CYP1A2 ontogeny. CYP2C8 and 1A2 protein levels were measured by quantitative Western blotting using liver microsomal samples prepared from 222 subjects with ages ranging from 8 weeks gestation to 18 years after birth. The median CYP2C8 expression was significantly greater among samples from subjects older than 35 postnatal days (n = 122) compared with fetal samples and those from very young infants (fetal to 35 days postnatal, n = 100) (0.00 vs. 13.38 pmol/mg microsomal protein; p < 0.0001). In contrast, the median CYP1A2 expression was significantly greater after 15 months postnatal age (n = 55) than in fetal and younger postnatal samples (fetal to 15 months postnatal, n = 167) (0.0167 vs. 2.354 pmol/mg microsomal protein; p < 0.0001). CYP2C8, but not CYP1A2, protein levels significantly correlated with those of CYP2C9, CYP2C19, and CYP3A4 (p < 0.001), consistent with CYP2C8 and CYP1A2 ontogeny probably being controlled by different mechanisms. This study provides key data for the physiologically based pharmacokinetic model-based prediction of age-dependent pyrethroid metabolism, which will be used for IVIVE to support pyrethroid risk assessment for early life stages.

Role of Chromatin Structural Changes in Regulating Human CYP3A Ontogeny.

Variability in drug-metabolizing enzyme developmental trajectories contributes to interindividual differences in susceptibility to chemical toxicity and adverse drug reactions, particularly in the first years of life. Factors linked to these interindividual differences are largely unknown, but molecular mechanisms regulating ontogeny are likely involved. To evaluate chromatin structure dynamics as a likely contributing mechanism, age-dependent changes in modified and variant histone occupancy were evaluated within known CYP3A4 and 3A7 regulatory domains. Chromatin immunoprecipitation using fetal or postnatal human hepatocyte chromatin pools followed by quantitative polymerase chain reaction DNA amplification was used to determine relative chromatin occupancy by modified and variant histones. Chromatin structure representing a poised transcriptional state (bivalent chromatin), indicated by the occupancy by modified histones associated with both active and repressed transcription, was observed for CYP3A4 and most 3A7 regulatory regions in both postnatal and fetal livers. However, the CYP3A4 regulatory regions had significantly greater occupancy by modified histones associated with repressed transcription in the fetal liver. Conversely, some modified histones associated with active transcription exhibited greater occupancy in the postnatal liver. CYP3A7 regulatory regions also had significantly greater occupancy by modified histones associated with repressed transcription in the fetus. The observed occupancy by modified histones is consistent with chromatin structural dynamics contributing to CYP3A4 ontogeny, although the data are less conclusive regarding CYP3A7. Interpretation of the latter data may be confounded by cell-type heterogeneity in the fetal liver.

Age-Dependent Human Hepatic Carboxylesterase 1 (CES1) and Carboxylesterase 2 (CES2) Postnatal Ontogeny.

Human hepatic carboxylesterase 1 and 2 (CES1 and CES2) are important for the disposition of ester- and amide-bond-containing pharmaceuticals and environmental chemicals. CES1 and CES2 ontogeny has not been well characterized, causing difficulty in addressing concerns regarding juvenile sensitivity to adverse outcomes associated with exposure to certain substrates. To characterize postnatal human hepatic CES1 and CES2 expression, microsomal and cytosolic fractions were prepared using liver samples from subjects without liver disease (N = 165, aged 1 day to 18 years). Proteins were fractionated, detected, and quantitated by Western blotting. Median microsomal CES1 was lower among samples from subjects younger than 3 weeks (n = 36) compared with the rest of the population (n = 126; 6.27 vs. 17.5 pmol/mg microsomal protein, respectively; P < 0.001; Kruskal-Wallis test). Median cytosolic CES1 expression was lowest among samples from individuals between birth and 3 weeks of age (n = 36), markedly greater among those aged 3 weeks to 6 years (n = 90), and modestly greater still among those older than 6 years (n = 36; median values = 4.7, 15.8, and 16.6 pmol/mg cytosolic protein, respectively; P values < 0.001 and 0.05, respectively; Kruskal-Wallis test). Median microsomal CES2 expression increased across the same three age groups with median values of 1.8, 2.9, and 4.2 pmol/mg microsomal protein, respectively (P < 0.001, both). For cytosolic CES2, only the youngest age group differed from the two older groups (P < 0.001; median values = 1.29, 1.93, 2.0, respectively). These data suggest that infants younger than 3 weeks of age would exhibit significantly lower CES1- and CES2-dependent metabolic clearance compared with older individuals.

Human hepatic UGT2B15 developmental expression.

Human hepatic UGT2B15 developmental expression changes may alter the metabolism of important drugs and toxicants such as bisphenol A (BPA). Previously, UGT2B15 ontogeny knowledge consisted of transcript data, a dubious surrogate for protein expression. Herein, UGT2B15 protein content was determined in human hepatic microsomes (n = 236, 8 weeks gestation to 18 years). The impact of a common, functional single nucleotide polymorphism (g.253G>T), present in UGT2B15*2 and *5 alleles, was also tested. UGT2B15 expression began during late fetal life, at about 18% of mature values (medians = 48, 267 pmoles/mg of microsomal protein, respectively; p < 0.001). UGT2B15 neonatal (n = 39) and late fetal (≥28 weeks, n = 10) content was similar, but lower than that of infants between 3 and 15 weeks age (n = 46; medians = 38, 48, 404 pmoles/mg microsomal protein, respectively; p < 0.001). Values for the latter group were higher compared with the remaining age group (15 weeks to 18 years; n = 82, p < 0.001). UGT2B15 expression varied 31-fold across the entire sample, and within groups, ranged from 4- to 27-fold. Among postnatal samples, age group, the presence of g.253T and male gender were each significantly associated with greater UGT2B15 expression (p < 0.001, <0.01, and <0.05, respectively; stepwise linear regression). In summary, hepatic UGT2B15 protein onset begins in late gestation; however, the greatest rate of change occurs during the first few weeks after birth. We speculate that the fetus and neonate may have lower clearance of some UGT2B15 substrates, such as BPA, compared with older individuals.

The effect of CYP2D6 polymorphisms on the response to pain treatment for pediatric sickle cell pain crisis.

OBJECTIVES: To test the hypothesis that children taking hydroxyurea who fail codeine therapy have an increase in reduced-functioning cytochrome P450 2D6 (CYP2D6) alleles. STUDY DESIGN: Children with sickle cell disease presenting to an emergency department with a pain crisis unresponsive to codeine were genotyped. The proportion of children with reduced-functioning alleles and CYP2D6 enzyme activity scores < or = 1.5, were compared, by chi2 analysis, in children taking hydroxyurea and those with mild disease. RESULTS: Of the 73 children completing the study, 42 had reduced-functioning alleles; 82% of the 27 children taking hydroxyurea had reduced-functioning alleles, versus 47% of 36 those with mild disease (P < .05). Activity scores were decreased in 78% of the children taking hydroxyurea and in 44% of those with mild disease (P < .05). The odds ratios of children taking hydroxyurea were 4.9 (95% confidence interval [CI] = 1.5 to 15.9) for having reduced-functioning alleles, and 4.4 (95% CI = 1.4 to 13.4) for having a low activity score. CONCLUSIONS: Failing codeine therapy for a pain crisis while taking hydroxyurea is associated with an increase in reduced-functioning CYP2D6 alleles. We recommend genetic analysis or trial of a non-CYP2D6 analgesic for these children.

Identification and functional analysis of a novel human CYP2E1 far upstream enhancer.

Both transcriptional and post-transcriptional CYP2E1 regulatory mechanisms are known, resulting in 20-fold or greater variation in CYP2E1 expression. To evaluate functional regulatory elements controlling transcription, CYP2E1 promoter constructs were used to make adenovirus vectors containing CYP2E1 promoter-driven luciferase reporters for analyses in both primary human hepatocytes and HepG2 cells. A 1.2-kilobase pair portion of the CYP2E1 promoter was associated with 5- to 10-fold greater luciferase activity. This upstream region contained five direct repeats of 59 base pairs (bp) that increased thymidine kinase-driven luciferase reporter activity in HepG2 cells more than 5-fold, regardless of orientation. Electrophoretic mobility shift assays (EMSAs) identified sequence-specific nuclear protein binding to the 59-bp repeats that was dependent on a 17-bp sequence containing a canonical GATA binding site (WGATAR). Competitive and supershift EMSA identified the participation of GATA4, another GATA family member or GATA-like factor, and a third factor unrelated to the GATA family. Involvement of the tricho-rhino-phalangeal syndrome-1 factor, which also binds a GATA sequence, was eliminated. Rather, competitive EMSA using known binding sequences for the orphan nuclear receptors, steroidogenic factor-1 (or NR5A1), and fetoprotein transcription factor (or NR5A2) implicated an NR5A member in binding a sequence overlapping the canonical GATA. Chromatin immunoprecipitation assay demonstrated in vivo binding of NR5A2 to the enhancer sequence in human hepatocytes. The enhancer sequence is conserved within the human population but seems species-specific. The identification of this novel enhancer and its putative mechanism adds to the complexities of human CYP2E1 regulation.

Pharmacogenomics and the future of drug therapy.

With the completion of the human genome project, many investigators are striving to translate the resulting wealth of new information into new and improved clinical practices. Pharmacogenomics represents one of the most promising of these applications for adult- and pediatric-based therapies. This article provides a historical perspective, but most importantly, uses this background to illustrate important principles of the field. The application of pharmacogenomics to asthma therapy is presented as an example of the current status of pharmacogenomics as it is being applied to an important pediatric health problem. Finally, a discussion of future promises and challenges to the application of pharmacogenomics is presented, including economic and ethical issues.

Population-based analysis of methadone distribution and metabolism using an age-dependent physiologically based pharmacokinetic model.

Limited pharmacokinetic (PK) and pharmacodynamic (PD) data are available to use in methadone dosing recommendations in pediatric patients for either opioid abstinence or analgesia. Considering the extreme inter-individual variability of absorption and metabolism of methadone, population-based PK would be useful to provide insight into the relationship between dose, blood concentrations, and clinical effects of methadone. To address this need, an age-dependent physiologically based pharmacokinetic (PBPK) model has been constructed to systematically study methadone metabolism and PK. The model will facilitate the design of cost-effective studies that will evaluate methadone PK and PD relationships, and may be useful to guide methadone dosing in children. The PBPK model, which includes whole-body multi-organ distribution, plasma protein binding, metabolism, and clearance, is parameterized based on a database of pediatric PK parameters and data collected from clinical experiments. The model is further tailored and verified based on PK data from individual adults, then scaled appropriately to apply to children aged 0-24 months. Based on measured variability in CYP3A enzyme expression levels and plasma orosomucoid (ORM2) concentrations, a Monte-Carlo-based simulation of methadone kinetics in a pediatric population was performed. The simulation predicts extreme variability in plasma concentrations and clearance kinetics for methadone in the pediatric population, based on standard dosing protocols. In addition, it is shown that when doses are designed for individuals based on prior protein expression information, inter-individual variability in methadone kinetics may be greatly reduced.

Discovery of novel flavin-containing monooxygenase 3 (FMO3) single nucleotide polymorphisms and functional analysis of upstream haplotype variants.

The flavin-containing monooxygenases (FMOs) are important for xenobiotic metabolism. FMO3, the predominant FMO enzyme in human adult liver, exhibits significant interindividual variation that is poorly understood. This study was designed to identify common FMO3 genetic variants and determine their potential for contributing to interindividual differences in FMO3 expression. FMO3 single nucleotide polymorphism (SNP) discovery was accomplished by resequencing DNA samples from the Coriell Polymorphism Discovery Resource. Population-specific SNP frequencies were determined by multiplexed, single-base extension using DNA from 201 Hispanic American (Mexican descent), 201 African American, and 200 White (northern European descent) subjects. Haplotypes were inferred and population frequencies estimated using PHASE version 2.1. Multiple site-directed mutagenesis was used to introduce inferred upstream haplotypes into an FMO3/luciferase construct for functional analysis in HepG2 cells. Sequence analysis revealed seven FMO3 upstream SNPs, 11 exon SNPs, and 22 intron SNPs. Five of the latter fell within consensus splice sites. A g.72G>T variant (E24D) is predicted to impact the structure of the Rossmann fold involved in FAD binding, whereas a g.11177C>A variant (N61K) is predicted to disrupt the secondary structure of a conserved membrane interaction domain. Seven common (>1%) promoter region haplotypes were inferred in one or more of the study populations that differed in estimated frequency among the groups. Haplotype 2 resulted in an 8-fold increase in promoter activity, whereas haplotypes 8 and 15 exhibited a near complete loss of activity. In conclusion, FMO3 promoter haplotype variants modulate gene function and probably contribute to interindividual differences in FMO3 expression.

Alcohol dehydrogenase 2*3 affects alterations in offspring facial morphology associated with maternal ethanol intake in pregnancy.

BACKGROUND: Ethanol intake during pregnancy alters offspring facial morphology. However, significant variation that may be due to genetic diversity in ethanol metabolizing enzymes occurs. The alcohol dehydrogenase 1B*3 (ADH1B*3) allele is protective for offspring developmental outcome after maternal alcohol drinking in pregnancy and may explain the spectrum of facial morphology. METHODS: Faces of infants with known ADH1B genotype, whose mothers' ADH1B genotypes and ethanol intake were determined during pregnancy, were photographed using uniform procedures. Photographs were scanned and the inner canthal distance, palpebral fissure length, and distance from the bridge of the nose to the bottom of the upper lip were measured by an investigator who was blinded to genotype and ethanol exposure. RESULTS: Among 247 photographed infants, each facial measurement varied >2-fold. Median absolute ethanol daily intake was 0.5 oz among mothers who reported drinking in the periconceptional period (N = 173) and 0.17 oz among mothers who reported drinking before the first prenatal visit (N = 62). Controlling for the amount of maternal ethanol intake just before the first prenatal visit and infant sex, the three-way interaction among the absence of a maternal and offspring ADH1B*3 allele and the presence of ethanol consumption just before the first prenatal visit was associated with smaller facial measurements (p = 0.002, MANCOVA). CONCLUSIONS: These are the first observations of a significant gene-environment interaction explaining variation in facial morphology associated with ethanol use in pregnancy. This positive effect of ADH1B*3 is consistent with its known positive effect on offspring birth weight and developmental outcome after in utero ethanol exposure.

Proximity of residence to trichloroethylene-emitting sites and increased risk of offspring congenital heart defects among older women.

BACKGROUND: Previous studies suggest that trichloroethylene (TCE) is a selective cardiac teratogen. We tested the hypothesis that the odds of maternal residence close to TCE-emitting sites would be greater among infants with congenital heart defects (CHDs) than among infants without CHDs. METHODS: We conducted a case-control study of 4025 infants, identified from hospital and birth records, born from 1997 to 1999 to Milwaukee, Wisconsin mothers. A geographic information system was used to calculate distances between maternal residences and TCE sites. We used classification tree analysis to determine appropriate values by which to dichotomously categorize mothers by TCE exposure (exposed: residence within 1.32 miles of at least one TCE site) and age (older: >/=38 years), and logistic regression to test for CHD risk factors. RESULTS: The proportion of mothers who were both older and had presumed TCE exposure was more than six-fold greater among case infants than among control infants (3.3% [8/245] versus 0.5% [19/3780]). When adjusted for other variables, CHD risk was over three-fold greater among infants of older, exposed mothers compared to infants of older, nonexposed mothers (adjusted OR, 3.2; 95% CI, 1.2-8.7). Older maternal age, alcohol use, chronic hypertension, and preexisting diabetes were each associated with CHDs (adjusted ORs, 1.9, 2.1, 2.8, 4.1; 95% CIs, 1.1-3.5, 1.1-4.2, 1.2-6.7, 1.5-11.2, respectively), but residence close to TCE sites alone was not. CONCLUSIONS: Our findings suggest that maternal age and TCE exposure interact to increase CHD risk, although the mechanism by which this occurs is unknown. A prospective study is underway to confirm this finding.

Applicability of the principles of developmental pharmacology to the study of environmental toxicants.

Although nontherapeutic xenobiotics represent the vast majority of environmental exposures during childhood, study of these compounds in children has lagged behind drug studies. Some useful extrapolation can be made from the latter, however. An increased impetus for pediatric pharmacology studies resulted from evidence of shortcomings in algorithmic approaches to dosing and the recognition of differing efficacy and toxicity in children compared with adults. With some drugs, developmental differences resulted in increased toxicity or failed efficacy; however, in others, decreased toxicity has been demonstrated. Thus, pediatric patients may not be classified arbitrarily as a susceptible population but certainly a different one compared with adults. Better designed pediatric pharmacology studies use well-documented, nonlinear changes in body composition across childhood, as well as knowledge about the impact of physical growth, mediated by complex hormonal changes. Developmental differences in all components of drug disposition, including absorption, distribution, metabolism, and excretion, have been characterized. Of these, the ontogeny of metabolism, particularly tissue-specific metabolism, is the most complex. Many knowledge gaps persist within developmental pharmacology; however, recent Food and Drug Administration regulatory action likely will ensure continued accumulation of pediatric therapeutic data. Although these data can provide important a priori information for improved environmental study design, evaluation-specific toxicant disposition by pediatric patients is clearly needed.

Excess birth prevalence of Hypoplastic Left Heart syndrome in eastern Wisconsin for birth cohorts 1997-1999.

BACKGROUND: Hypoplastic Left Heart syndrome (HLHS) is a group of cardiac malformations involving underdevelopment of the left heart with an inability to maintain systemic circulation. Because of a clinical impression of excess HLHS prevalence, we completed a medical record review of cases born from 1997 through 1999 who were Wisconsin residents and seen at the Children's Hospital of Wisconsin (CHW). METHODS: Cases were identified either in the CHW medical records database or the Division of Pediatric Cardiology database and confirmed by echocardiogram, catheterization, surgery, or autopsy. U.S. and international surveillance systems were used to estimate population risk. Rates per 10,000 births were computed for eastern Wisconsin and four regions within this portion of the state, and compared to the estimated population risk. The same methods were used to evaluate whether rates for tetralogy of Fallot and transposition of the great arteries were elevated. RESULTS: A total of 61 cases were ascertained yielding a birth prevalence of 3.7 per 10,000 births, which was greater than the estimated population risk of 2.79. The rate for the southeast region also exceeded the expected rate. The most urban and industrialized areas had the highest rates. Rates for the other two diagnoses evaluated were not different from estimated population risks. CONCLUSIONS: Eastern Wisconsin, particularly the urban southeast region, had elevated rates of HLHS. Because of the geographic clustering of high rates, environmental factors may be associated with this finding.

Developmental expression of human hepatic CYP2C9 and CYP2C19.

The CYP2C subfamily is responsible for metabolizing many important drugs and accounts for about 20% of the cytochrome p450 in adult liver. To determine developmental expression patterns, liver microsomal CYP2C9 and -2C19 were measured (n = 237; ages, 8 weeks gestation-18 years) by Western blotting and with diclofenac or mephenytoin, respectively, as probe substrates. CYP2C9-specific content and catalytic activity were consistent with expression at 1 to 2% of mature values (i.e., specific content, 18.3 pmol/mg protein and n = 79; specific activity, 549.5 pmol/mg/min and n = 72) during the first trimester, with progressive increases during the second and third trimesters to levels approximately 30% of mature values. From birth to 5 months, CYP2C9 protein values varied 35-fold and were significantly higher than those observed during the late fetal period, with 51% of samples exhibiting values commensurate with mature levels. Less variable CYP2C9 protein and activity values were observed between 5 months and 18 years. CYP2C19 protein and catalytic activities that were 12 to 15% of mature values (i.e., specific content, 14.6 pmol/mg and n = 20; specific activity, 18.5 pmol/mg/min and n = 19) were observed as early as 8 weeks of gestation and were similar throughout the prenatal period. CYP2C19 expression did not change at birth, increased linearly over the first 5 postnatal months, and varied 21-fold from 5 months to 10 years. Adult CYP2C19 protein and activity values were observed in samples older than 10 years. The ontogeny of CYP2C9 and -2C19 were dissimilar among both fetal and 0- to 5-months postnatal samples, implying different developmental regulatory mechanisms.

Human hepatic CYP2E1 expression during development.

Human hepatic CYP2E1 expression developmental changes likely have an impact on the effects of xenobiotics metabolized by the encoded enzyme. To resolve previous conflicting results, CYP2E1 content was determined in human hepatic microsomes from samples spanning fetal (n = 73, 8-37 weeks) and postnatal (n = 165, 1 day-18 years) ages. Measurable immunodetectable CYP2E1 was seen in 18 of 49 second-trimester (93-186 gestational days) and 12 of 15 third-trimester (>186 days) fetal samples (medians = 0.35 and 6.7 pmol/mg microsomal protein, respectively). CYP2E1 in neonatal samples was low and less than that of infants 31 to 90 days of age, which was less than that of older infants, children, and young adults [median (range) = 8.8 (0-70); 23.8 (10-43); 41.4 (18-95) pmol/mg microsomal protein, respectively; each P < 0.001, analysis of variance, post hoc]. Among those older than 90 days of age, CYP2E1 content was similar. A 4-fold or greater intersubject variation was observed among samples from each age group, with the greatest variation, 80-fold, seen among neonatal samples. Among subjects of known gestational and postnatal age (n = 29) increasing protein content was associated with increasing postnatal age (P < 0.001, linear regression), but only equivocally with increasing gestational age (P = 0.07). Individuals from the third trimester through 90 days postnatal age with one or more CYP2E1*1D alleles had lower CYP2E1 protein content than similar-aged subjects who were homozygous CYP2E1*1C. In summary, CYP2E1 was clearly expressed in human fetal liver. Furthermore, the postnatal data suggest that infants less than 90 days old would have decreased clearance of CYP2E1 substrates compared with older infants, children, and adults.

Genetic variability at the human FMO1 locus: significance of a basal promoter yin yang 1 element polymorphism (FMO1*6).

The flavin-containing monooxygenases (FMOs) are important for the disposition of a variety of toxicants, therapeutics, and dietary components. Although FMO1 is the dominant isoform in fetal liver and adult kidney and intestine and despite up to a 10-fold intersubject variation in expression, a paucity of information is available on FMO1 genetic variability. To address this issue, 24 samples from the Coriell DNA Polymorphism Discovery Resource Panel were sequenced revealing 10 common single nucleotide polymorphisms (SNPs): four located upstream of the structural gene; three within exonic sequences; one within the intron 1 splice donor site; and two with the 3'-untranslated region. Six of these variants are novel. Compared with other FMO loci within the chromosome 1q23-25 cluster, FMO1 seems more highly conserved. Of the identified FMO1 SNPs, only a C>A transversion 9536 base pairs upstream of the exon 2 ATG start codon (g.-9536C>A) would likely affect function, because it lies within the conserved core binding sequence for the yin yang 1 (YY1) transcription factor. Electrophoretic mobility shift assays demonstrated that the g.-9536C>A transversion eliminated YY1 binding. Furthermore, data from transient expression assays in HepG2 cells suggested this SNP could account for a 2- to 3-fold loss of FMO1 promoter activity. Genotype analysis revealed a g.-9,536A allele (FMO1*6) frequency of 13 and 11% in African- and northern European-Americans, respectively, but a significantly higher frequency of 30% in Hispanic-Americans. Thus, the FMO1*6 variant may account for some of the observed interindividual variation in FMO1 expression.

Trichloroethylene decreases heat shock protein 90 interactions with endothelial nitric oxide synthase: implications for endothelial cell proliferation.

Trichloroetheylene (TRI) is an environmental pollutant that has been linked to congenital heart defects (CHD). Endothelial nitric oxide synthase (eNOS) generation of nitric oxide (NO) plays an important role in endothelial cell proliferation, which is considered essential for normal blood vessel growth and development. We hypothesized that TRI alters the balance of NO and superoxide anion (O2-) to impair endothelial cell proliferation. Proliferating endothelial cells were pretreated with TRI (5 microM) and then stimulated with the calcium ionophore, A23187 (5 microM), to determine changes in endothelial cell and eNOS function with respect to NO and O2- generation. Immunoblots of eNOS, phospho-eNOS at serine 1179 (S1179), and the levels of associated heat shock protein 90 (hsp90) were used to define the activation state of eNOS. The effects of TRI (0.05-100 microM) on vascular endothelial growth factor (VEGF, 0.58 nM) induced endothelial cell proliferation were determined from cell counts. TRI decreased A23187-stimulated nitrite + nitrate production from 1.99 +/- 0.90 to 0.89 +/- 0.51 pmol/mg protein (p < 0.05; n = 6). In controls, Lomega-nitroargininemethylester (L-NAME) increased A23187-stimulated O2- production from 0.130 +/- 0.089 to 0.214 +/- 0.071 nmol/min/mg protein (p < 0.05; n = 5). In TRI-treated cultures, however, L-NAME decreased A23187-stimulated O2- production from 0.399 +/- 0.121 to 0.199 +/- 0.055 nmol/min/mg protein (p < 0.05; n = 5). TRI decreased hsp90 associated with eNOS by 46.7% and inhibited VEGF-stimulated endothelial cell proliferation by 12 to 35%. These data show that TRI alters hsp90 interactions with eNOS and induces eNOS to shift from NO to O2- generation. Our findings provide new insight into how TRI alters endothelial and eNOS function to impair VEGF-stimulated endothelial proliferation. Such changes in endothelial function may play an important role in the development of congenital heart defects.

Genotyping and site-directed mutagenesis of a cytochrome P450 meander Pro-X-Arg motif critical to CYP4B1 catalysis.

CYP4B1 isoforms from rodents and other common laboratory animals are involved in the bioactivation of a range of protoxins, including 2-aminofluorene, 4-ipomeanol, and valproic acid. However, an earlier study provided evidence for a human allele encoding a nonfunctional CYP4B1 enzyme due to a Pro427Ser transversion in the meander region of the protein. In the present study, the CYP4B1 gene from several racial groups, Caucasians, African-Americans, and Hispanics, and from six nonhuman primate species was genotyped using a PCR-Hinf1 restriction enzyme fragment length polymorphism assay or by direct sequencing. All human populations examined were found to possess only the Ser allele at codon 427 ((1279)TCT) and all of the nonhuman primate species possessed only the Pro (CCT) allele. Therefore, an inactivating (1279)C-->T mutation in the human CYP4B1 gene likely arose following divergence of the Homo and Pan clades. Amino acid sequence alignments revealed further that this key Pro residue is located two amino acid residues N-terminal to the distal Arg of a Glu-Arg-Arg triad thought to participate in heme binding and/or redox partner interactions. Mutation of the corresponding Arg424 residue in rabbit CYP4B1 to Leu, but not His, resulted in a loss of lauric acid hydroxylase activity and ability to generate a reduced-CO binding spectrum. These data provide additional evidence for the importance of this meander region Pro-X-Arg motif in CYP4B1 heme binding and catalytic function.

Completeness of state administrative databases for surveillance of congenital heart disease.

BACKGROUND: Tracking birth prevalence of cardiac defects is essential to determining time and space clusters, and identifying potential associated factors. Resource limitations on state birth defects surveillance programs sometimes require that databases already available be used for ascertaining such defects. This study evaluated the data quality of state administrative databases for ascertaining congenital heart defects (CHD) and specific diagnoses of CHD. METHODS: Children's Hospital of Wisconsin (CHW) medical records for infants born 1997-1999 and treated for CHD (n = 373) were abstracted and each case assigned CHD diagnoses based on definitive diagnostic reports (echocardiograms, catheterizations, surgical or autopsy reports). These data were linked to state birth and death records, and birth and postnatal (< 1 year of age) hospital discharge summaries at the Wisconsin Bureau of Health Information (WBHI). Presence of any code/checkbox indicating CHD (generic CHD) and exact matches to abstracted diagnoses were evaluated. RESULTS: Fifty-eight percent of cases with generic CHD were identified by state databases. Postnatal hospital discharge summaries identified 48%, birth hospital discharge summaries 27%, birth certificates 9% and death records 4% of these cases. Exact matches were found for 52% of 633 specific diagnoses. Postnatal hospital discharge summaries provided most matches. CONCLUSION: State databases identified 60% of generic CHD and exactly matched about half of specific CHD diagnoses. The postnatal hospital discharge summaries performed best in both in identifying generic CHD and matching specific CHD diagnoses. Vital records had limited value in ascertaining CHD.

The ontogeny of human drug-metabolizing enzymes: phase I oxidative enzymes.

Although some patterns are beginning to emerge, our knowledge of human phase I drug-metabolizing enzyme developmental expression remains far from complete. Expression has been observed as early as organogenesis, but this appears restricted to a few enzymes. At least two of the enzyme families that are expressed in the fetal liver exhibit a temporal switch in the immediate perinatal period (e.g., CYP3A7 to CYP3A4/3A5 and FMO1 to FMO3), whereas others show a progressive change in isoform expression through gestation (e.g., the class I alcohol dehydrogenases). Many of the phase I drug-metabolizing enzyme exhibit dynamic perinatal expression changes that are regulated primarily by mechanisms linked to birth and secondarily to maturity. A few of these enzymes are not detectable until well after birth, suggesting that birth is necessary but not sufficient for the onset of expression (e.g., CYP1A2). Tissue-specific expression adds to the complexity during ontogeny. For example, CYP3A7 expression is restricted to the fetal liver. However, with few exceptions, complete temporal relationship information during development is not known. Furthermore, most studies have concentrated on hepatic expression and much less is known about extrahepatic developmental events.