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Endomyocardial biopsy (EMB) is the current standard for rejection surveillance in heart transplant recipients. The quantification of donor-specific cell-free DNA (cfDNA) may be an appropriate biomarker for non-invasive rejection surveillance. A multicenter prospective blinded study (DNA-Based Transplant Rejection Test, DTRT) investigated the value of donor fraction (DF), defined as the ratio of cfDNA specific to the transplanted organ to the total amount of cfDNA present in a blood sample.A total of 241 heart transplant patients were recruited from 7 centers. Age at transplant ranged from 8 days to 73 years, with 146 subjects18 years and 95 ≥18 years. All the patients were followed for at least 1 year, with blood samples drawn at routine and for-cause biopsies. A total of 624 biopsy-paired samples were included for analysis through a commercially available cfDNA assay (myTAIDF in acute cellular rejection (ACR) 1R/2R (n = 15) was higher than ACR 0R (n = 42) (p = 0.02); DF in antibody-mediated rejection pAMR1 (n = 8) and pAMR2 (n = 12) (p = 0.05) were higher than pAMR0 (n = 466) (p = 0.04 and p = 0.05 respectively). An optimal DF threshold was determined by the use of an ROC analysis, which ruled out the presence of either ACR or antibody-mediated rejection.The cell-free DNA DF holds promise as a non-invasive diagnostic test to rule out acute rejection in both adult and pediatric heart transplant populations.

Heart transplantation is a well‐established therapy for end‐stage heart failure in children and young adults. The highest risk of graft loss occurs in the first 60 days post‐transplant. Donor fraction of cell‐free DNA is a highly sensitive marker of graft injury. Changes in cell‐free DNA levels have not previously been studied in depth in patients early after heart transplant. A prospective study was conducted among heart transplant recipients at a single pediatric heart center. Blood samples were collected from children and young adult transplant patients at three time points within 10 days of transplantation. DF and total cell‐free DNA levels were measured using a targeted method (myTAIHEART). In 17 patients with serial post‐transplant samples, DF peaks in the first 2 days after transplant (3.5%, [1.9‐10]%) and then declines toward baseline (0.27%, [0.19‐0.52]%) by 6‐9 days. There were 4 deaths in the first year among the 10 patients with complete sample sets, and 3 out of 4 who died had a late rise or blunted decline in donor fraction. Patients who died trended toward an elevated total cell‐free DNA at 1 week (41.5, [34‐65] vs 13.6, [6.2‐22] P = .07). Donor fraction peaks early after heart transplant and then declines toward baseline. Patients without sustained decline in donor fraction and/or elevated total cell‐free DNA at 1 week may have worse outcomes.

Hypoplastic left heart syndrome (HLHS) is a clinically and anatomically severe form of congenital heart disease (CHD). Although prior studies suggest that HLHS has a complex genetic inheritance, its etiology remains largely unknown. The goal of this study was to characterize a risk gene in HLHS and its effect on HLHS etiology and outcome. We performed next-generation sequencing on a multigenerational family with a high prevalence of CHD/HLHS, identifying a rare variant in the α-myosin heavy chain ( MYH6) gene. A case-control study of 190 unrelated HLHS subjects was then performed and compared with the 1000 Genomes Project. Damaging MYH6 variants, including novel, missense, in-frame deletion, premature stop, de novo, and compound heterozygous variants, were significantly enriched in HLHS cases ( P < 1 × 10−5). Clinical outcomes analysis showed reduced transplant-free survival in HLHS subjects with damaging MYH6 variants ( P < 1 × 10−2). Transcriptome and protein expression analyses with cardiac tissue revealed differential expression of cardiac contractility genes, notably upregulation of the β-myosin heavy chain ( MYH7) gene in subjects with MYH6 variants ( P < 1 × 10−3). We subsequently used patient-specific induced pluripotent stem cells (iPSCs) to model HLHS in vitro. Early stages of in vitro cardiomyogenesis in iPSCs derived from two unrelated HLHS families mimicked the increased expression of MYH7 observed in vivo ( P < 1 × 10−2), while revealing defective cardiomyogenic differentiation. Rare, damaging variants in MYH6 are enriched in HLHS, affect molecular expression of contractility genes, and are predictive of poor outcome. These findings indicate that the etiology of MYH6-associated HLHS can be informed using iPSCs and suggest utility in future clinical applications.

The clinical significance of copy number variants (CNVs) in congenital heart disease (CHD) continues to be a challenge. Although CNVs including genes can confer disease risk, relationships between gene dosage and phenotype are still being defined. Our goal was to perform a quantitative analysis of CNVs involving 100 well-defined CHD risk genes identified through previously published human association studies in subjects with anatomically defined cardiac malformations. A novel analytical approach permitting CNV gene frequency “spectra” to be computed over prespecified regions to determine phenotype-gene dosage relationships was employed. CNVs in subjects with CHD ( n = 945), subphenotyped into 40 groups and verified in accordance with the European Paediatric Cardiac Code, were compared with two control groups, a disease-free cohort ( n = 2,026) and a population with coronary artery disease ( n = 880). Gains (≥200 kb) and losses (≥100 kb) were determined over 100 CHD risk genes and compared using a Barnard exact test. Six subphenotypes showed significant enrichment ( P ≤ 0.05), including aortic stenosis (valvar), atrioventricular canal (partial), atrioventricular septal defect with tetralogy of Fallot, subaortic stenosis, tetralogy of Fallot, and truncus arteriosus. Furthermore, CNV gene frequency spectra were enriched ( P ≤ 0.05) for losses at: FKBP6, ELN, GTF2IRD1, GATA4, CRKL, TBX1, ATRX, GPC3, BCOR, ZIC3, FLNA and MID1; and gains at: PRKAB2, FMO5, CHD1L, BCL9, ACP6, GJA5, HRAS, GATA6 and RUNX1. Of CHD subjects, 14% had causal chromosomal abnormalities, and 4.3% had likely causal (significantly enriched), large, rare CNVs. CNV frequency spectra combined with precision phenotyping may lead to increased molecular understanding of etiologic pathways.

To the Editor: Approximately 20,000 cardiac transplant recipients currently reside in the United States. Rejection remains a major cause of graft failure and requires lifelong surveillance. The current gold standard for monitoring rejection is catheter-based endomyocardial biopsy (EMB), which is

EtOH consumption significantly impaired anabolic rebuilding of bone after lactation. Lower BMD and BMC in EtOH-fed rats were associated with decreased bone formation in the proximal tibia, increased proportion of adipocytes, and increased expression of TNF-α. EtOH-induced skeletal deficits were prevented by treatment with either NAC or sTNFR1. These data suggest that postlactational anabolic rebuilding is influenced by EtOH consumption and may affect the long-term risk of osteopenia. Introduction: Despite significant loss of bone during lactation, BMD is restored by a powerful anabolic rebuilding process after weaning. A significant number of women resume alcohol consumption after weaning their offspring from breast feeding. The objectives of this study were to examine the consequences of chronic ethanol (EtOH) consumption on the postlactational rebuilding process and to investigate the underlying mechanisms by which EtOH mediates its detrimental effects. Materials and Methods: Female Sprague-Dawley rats (n = 7–9 per group) were fed EtOH-containing diets (13 g/kg/d) for 1, 2, or 4 wk after weaning of their offspring. Skeletal parameters in the proximal tibia were examined using pQCT, μCT, and histomorphometric techniques, and interventional studies were performed on the mechanistic roles of EtOH-induced oxidative stress and TNF-α. Results and Conclusions: EtOH consumption completely abolished the anabolic bone rebuilding that occurred after lactation. Decreased BMD and BMC were associated with decreased bone formation and not with increased osteoclast activity. Furthermore, EtOH-fed rats showed greater proportion of fat volume/bone volume and expression of adipocyte-specific genes. EtOH-induced skeletal effects were mitigated by the dietary antioxidant, N-acetyl cysteine or by blocking TNF-α signaling. These data suggest EtOH consumption in the period immediately postweaning may significantly impair the mother's skeletal health and lead to long-term osteopenia.

To investigate the effects of sex hormones on ethanol (EtOH)-induced bone loss, female Sprague-Dawley rats were fed control or EtOH-containing diets (12 g/kg/day) by intragastric infusion. After 3 weeks, rats receiving EtOH had significant decreases in tibial trabecular and total bone mineral density, induction of receptor activator of nuclear factor-kappaB ligand (RANKL) mRNA expression, and enhanced bone resorption, all of which were prevented by treatment with 17beta-estradiol (E(2)). The addition of progesterone did not enhance the beneficial effect of E(2) alone. Consistent with our in vivo findings, EtOH stimulated RANKL mRNA expression in cultured primary osteoblasts, and this expression was blocked by 4-methylpyrazole. Acetaldehyde also induced RANKL expression. Class 1 alcohol dehydrogenase was found to be expressed and EtOH-inducible in cultured osteoblasts, whereas CYP2E1 was undetectable. We found that EtOH induced phosphorylation of extracellular signal-regulated kinase (ERK) and signal transducers and activators of transcription 3 (STAT3). E(2) and the mitogenactivated protein kinase kinase inhibitor 2'-amino-3'-methoxyflavone (PD98059) blocked ERK and STAT3 phosphorylation and blocked RANKL induction. Moreover, E(2) completely blocked EtOH-induced osteoclastogenesis in a primary osteoblast and osteoclast precursor coculture system. The E(2) effects were estrogen receptor-mediated. Therefore, E(2) prevents EtOH-induced bone loss by opposing the induction of RANKL mRNA in osteoblasts and ethanol-induced osteoclastogenesis, through opposing effects on sustained ERK signaling.

The identification of the epitopes recognized by autoantibodies against cytochrome P450s (CYPs) associated with drug-induced hepatotoxicity is difficult because of their conformational nature. In the present investigation, we used a novel approach based on the analysis of the whole molecule antigenic capacity following single amino acid substitutions to identify the conformational epitopes on CYP2E1. A molecular model of CYP2E1 was generated based on the CYP2C5 crystal structure, and potential motifs for amino acid exchanges were selected by computer simulation in the surface of alpha helices and beta sheets. Fourteen modified, apparently correctly folded CYP2E1 variants were produced in Escherichia coli and evaluated in immunoprecipitation experiments using sera with anti-CYP2E1 autoreactivity from 10 patients with halothane hepatitis and 12 patients with alcoholic liver disease. Ala substitution of Glu-248 and Lys-251 as well as of Lys-324, Lys-342, Lys-420, and Phe-421 severely decreased or abolished CYP2E1 recognition by the majority of both the halothane hepatitis and alcoholic liver disease sera, whereas the other substitutions had only minor effects. Based on the structural model, these substitutions identified two distinct epitopes on the CYP2E1 surface corresponding to the G-helix and an area formed by juxtaposition of the J' and K'' helices, respectively. The combined use of molecular modeling and single amino acid mutagenesis is thus a useful approach for the characterization of conformational epitopes recognized by autoantibodies.

Several-fold differences have been observed among patients in the biotransformation of cyclophosphamide. The aim of this study was to investigate the contribution of CYP2C9 and CYP2C19 and their polymorphisms to the variability of cyclophosphamide activation. The formation of 4-hydroxycyclophosphamide was studied in microsomes from a total of 32 different genotyped human livers, as well as in yeast microsomes expressing different genetic variants of CYP2C9 and CYP2C19. The kinetic data obtained in the yeast system revealed that the intrinsic clearance (Vmax/Km) of cyclophosphamide by CYP2C9.2 and CYP2C9.3 samples was approximately threefold lower than that by CYP2C9.1. However, in liver microsomes, there were no statistically significant differences in the intrinsic clearance of 4-hydroxycyclophosphamide formation between the group of seven CYP2C9*1/*1 livers and the remaining nine with one or two variant CYP2C9 alleles (P>0.7). We found a statistically significant correlation (r s=0.65, P=0.003) between 4-hydroxylation of cyclophosphamide and 5′-hydroxylation of R-omeprazole, a measure of CYP2C19 activity in human liver microsomes (n=19). No correlation was found between 4-hydroxylation of cyclophosphamide and the formation rate of hydroxycelecoxib, mainly catalysed by CYP2C9 (r s=0.17, P=0.55, n=32). In conclusion, based on the correlation with the formation of R-5′-hydroxyomeprazole, CYP2C19 may partly contribute to the bioactivation of cyclophosphamide in human liver microsomes, while the role of CYP2C9 appears minor.

Donor fraction (DF) of cell-free DNA (cf-DNA) has been proposed as a noninvasive surveillance method in heart transplantation. We present our pilot experience with a novel assay to quantify DF (%) with a clinically practical throughput and turnaround time. A blinded study was performed in which

Objective: The present study was carried out to identify the cytochrome P 450 enzyme(s) involved in the 6-hydroxylation and O-demethylation of melatonin. Methods: The formation kinetics of 6-hydroxymelatonin and N-acetylserotonin were determined using human liver microsomes and cDNA yeast-expressed human enzymes (CYP1A2, 2C9 and 2C19) over the substrate concentration range 1–1000 µM. Selective inhibitors and substrates of various cytochrome P 450 enzymes were also employed. Results: Fluvoxamine was a potent inhibitor of 6-hydroxymelatonin formation, giving 50±5% and 69±9% inhibition at concentrations of 1 µM and 10 µM, respectively, after incubation with 50 µM melatonin. Furafylline, sulphaphenazole and omeprazole used at low and high concentrations substantially inhibited both metabolic pathways. cDNA yeast-expressed CYP1A2, CYP2C9 and CYP2C19 catalysed the formation of the two metabolites, confirming the data obtained with specific inhibitors and substrates. Conclusions: Our results strongly suggest that 6-hydroxylation, the main metabolic pathway of melatonin, is mediated mainly, but not exclusively, by CYP1A2, the high-affinity enzyme involved in melatonin metabolism, confirming the observation that a single oral dose of fluvoxamine increases nocturnal serum melatonin levels in healthy subjects. Furthermore, the results indicate that there is a potential for interaction with drugs metabolised by CYP1A2 both at physiological levels and after oral administration of melatonin, while CYP2C19 and CYP2C9 are assumed to be less important.

1. The ability of various in vitro systems for CYP enzymes (computer modelling, human liver microsomes, precision-cut liver slices, hepatocytes in culture, recombinant enzymes) to predict various aspects of in vivo metabolism and kinetics of carbamazepine (CBZ) was investigated. 2. The study was part of the EUROCYP project that aimed to evaluate relevant human in vitro systems to study drug metabolism. 3. CBZ was given to the participating laboratories without disclosing its chemical nature. 4. The most important enzyme (CYP3A4) and metabolic route (10,11-epoxidation) were predicted by all the systems studied. 5. Minor enzymes and routes were predicted to a different extent by various systems. 6. Prediction of a clearance class, i.e. slow clearance, was correctly predicted by microsomes, slices, hepatocytes and recombinant enzymes (CYP3A4). 7. The 10,11-epoxidation of CBZ by the recombinant CYP3A4 was enhanced by the addition of exogenous cytochrome-b5, leading to a considerable over-prediction. 8. Induction potency of CBZ was predicted in cultured hepatocytes in which 7-ethoxycoumarin O-deethylase was used as an index activity. 9. It seems that for a principally CYP-metabolized substance such as CBZ, all liver-derived systems provide useful information for prediction of metabolic routes, rates and interactions.

Cytochrome P450 1B1 (CYP1B1) is a human extrahepatic P450 that activates procarinogens, metabolizes 17β-estradiol, and may well have a role in the pathogenesis of some forms of cancer. Besides rare deleterious mutations reported for the CYP1B1 gene, six single-nucleotide polymorphisms have been reported, of which four cause amino acid exchanges. We have expressed two of the common CYP1B1 alleles in yeast cells and mammalian COS-1 cells in order to functionally characterize the alleles with respect to kinetic properties and protein stability. The CYP1B1.2 variant contains the two linked amino acid substitutions R48G and A119S compared to CYP1B1.1. The kinetic parameters of two structurally unrelated CYP1B1 substrates for the two variants were examined. No kinetic differences were seen of 17β-estradiol hydroxylation activities between the two CYP1B1 variants and an only minor increase in the apparent Km for ethoxyresorufin was observed for CYP1B1.2. It therefore appears that they have very similar catalytic properties and the substitutions do not appear to alter CYP1B1 catalytic function. The two CYP1B1 variants were similarly stable when expressed in mammalian COS-1 cells, indicating that the substitutions have no effect on protein folding or stability. The combined results indicate that these two CYP1B1 variants show very similar properties with respect to catalytic activities and protein stability and do not alter CYP1B1 function.

A cDNA coding for rat cytochrome P450 2E1 was cloned into the multicopy vector pYeDP60 and expressed in haploid RSY6 and diploid RS112 yeast strains of Saccharomyces cerevisiae under control of the GAL10-CYC1 promoter. Spectral and catalytic properties of the expressed 2E1 were examined in whole cells or microsomes of both strains. The level of CYP2E1 obtained in RS112 (200 pmol/mg microsomal protein) was the highest among CYP2E1 produced in the various expression systems. The monooxygenase activity in the microsomes of both strains, measured as aniline hydroxylase, was found comparable to that of control rat hepatic microsomes. In a reconstituted system in the presence of exogenous rat P450 reductase, their activity increased about 10-fold. When exposed to the carcinogen NDMA, a known 2E1 substrate, the recombination frequency determined in the 2E1-expressing RS112 cells was enhanced, in a dose-dependent manner, up to 20-fold. The exposure of the same cells to the hepatotoxic solvents, N-methyl- and N-ethylformamide, resulted in an induction of recombination frequency, which was not observed in the void plasmid containing RS112 cells in the presence of S9 hepatic fractions from pyrazole-induced rats, as a specific exogenous metabolic activation system. These results demonstrate that the 2E1-expressing cells metabolize the two N-alkylformamides to genotoxic intermediates and, therefore, they provide an useful tool to study the bioactivation mechanism of potential P450 2E1 substrates.

In many black African populations, the capacity for CYP2D6-dependent drug metabolism is generally reduced. A specific variant of the CYP2D6 gene (CYP2D6*17) that carries three functional mutations (T107I, R296C, and S486T) has been found to be present in Zimbabwean subjects with impaired CYP2D6-dependent hydroxylase activity. To evaluate whether the CYP2D6*17 allele was the major cause behind the decreased rate of drug metabolism and to examine the role of the different mutations, CYP2D6 cDNAs containing all eight combinations of the mutations were created. Expression of the cDNAs in COS-1 cells revealed that the CYP2D6 17 enzyme displayed only 20% of the wild-type (CYP2D6 1) activity, whereas the T107I substitution on its own had no significant effect on enzyme function. Expression in yeast showed that the three possible single amino-acid mutant CYP2D6 variants all had properties similar to CYP2D6 1 when the kinetics of bufuralol hydroxylation was examined. However, enzymes containing both the T107I and R296C mutations exhibited a more than 5-fold higher K(m) for bufuralol than the wild-type enzyme, whereas the S486T mutation was of little importance. In contrast, when codeine was used as a substrate, the T107I substitution alone was sufficient to cause a significant increase in the apparent K(m), indicating a differential effect for this substitution depending on the CYP2D6 substrate. In conclusion, the CYP2D6*17 allele represents the first human cytochrome P450 polymorphic variant in which a combination of substitutions is required to alter the enzyme's catalytic properties and is the first case in which a decreased CYP2D6 activity, as monitored in vivo, has been documented to be caused by an enzyme with altered affinity for CYP2D6 substrates.

Objective: To determine the effect of shipping blood in Streck blood collection tubes (BCT) prior to processing on cell-free DNA (cf-DNA) levels. Methods: Blood was collected in ethylenediaminetetraacetic acid (EDTA) and BCT tubes from 10 pregnant women carrying male fetuses. One set of each tube for each subject was processed to plasma immediately (standard cf-DNA protocol), whereas the other set was shipped by air courier and then processed. DNA was extracted and total and fetal DNA concentrations were measured by TaqMan multiplexed quantitative real-time PCR. Results: No significant difference was observed in total cf-DNA in plasma between immediately processed EDTA (control) and immediately processed BCT samples. Moreover, no significant change in total cf-DNA was detected in plasma of BCT samples shipped at room temperature. Significant differences in total cf-DNA leading to a significantly decreased fetal fraction were found in shipped EDTA samples and BCT samples shipped at 4°C. Discussion: BCT tubes are suitable for shipping whole blood prior to processing with respect to cf-DNA levels. However, care should be taken to ensure that samples are not exposed to extreme temperatures during shipment. This finding will benefit the development and clinical application of noninvasive methods of fetal diagnosis that utilize cf-DNA.

Background Screening tests for Trisomy 21 (T21), also known as Down syndrome, are routinely performed for the majority of pregnant women. However, current tests rely on either evaluating non-specific markers, which lead to false negative and false positive results, or on invasive tests, which while highly accurate, are expensive and carry a risk of fetal loss. We outline a novel, rapid, highly sensitive, and targeted approach to non-invasively detect fetal T21 using maternal plasma DNA. Methods and Findings Highly heterozygous tandem Single Nucleotide Polymorphism (SNP) sequences on chromosome 21 were analyzed using High-Fidelity PCR and Cycling Temperature Capillary Electrophoresis (CTCE). This approach was used to blindly analyze plasma DNA obtained from peripheral blood from 40 high risk pregnant women, in adherence to a Medical College of Wisconsin Institutional Review Board approved protocol. Tandem SNP sequences were informative when the mother was heterozygous and a third paternal haplotype was present, permitting a quantitative comparison between the maternally inherited haplotype and the paternally inherited haplotype to infer fetal chromosomal dosage by calculating a Haplotype Ratio (HR). 27 subjects were assessable; 13 subjects were not informative due to either low DNA yield or were not informative at the tandem SNP sequences examined. All results were confirmed by a procedure (amniocentesis/CVS) or at postnatal follow-up. Twenty subjects were identified as carrying a disomy 21 fetus (with two copies of chromosome 21) and seven subjects were identified as carrying a T21 fetus. The sensitivity and the specificity of the assay was 100% when HR values lying between 3/5 and 5/3 were used as a threshold for normal subjects. Conclusions In summary, a targeted approach, based on calculation of Haplotype Ratios from tandem SNP sequences combined with a sensitive and quantitative DNA measurement technology can be used to accurately detect fetal T21 in maternal plasma when sufficient fetal DNA is present in maternal plasma., Wallace H. Coulter Foundation, Medical College of Wisconsin. Biotechnology and Bioengineering Center (Inducement Grant Fund), National Science Foundation (U.S.) (PFI grant NSF#0438604), Medical College of Wisconsin. Dept. of Surgery, Tandem Diagnostics (Firm)

We report an age-dependent increase in nonimmunohematopoietic cells (CD45 neg ) in regenerating muscle characterized by high stem-cell antigen (Sca-1) expression. In aged regenerating muscle, only 14% of these CD45 neg Sca-l pos cells express MyoD, whereas 82% of CD45 neg Sca-l pos cells are MyOD pos in young adult muscle. In vitro, CD45 neg MyoD neg Sca-l pos cells overexpress fibrosis-promoting genes, potentially controlled by Wnt2. The cells are proliferative, nonmyogenic, and nonadipogenic, and arise in clonally derived myoblast cultures from aged mice. MyoD neg Sca-l pos nonmyogenic cells also emerge in C2C12 myoblast cultures at late passage. Both in vitro and in vivo studies suggest that MyoD neg sca-l pos cells from aged muscle are more susceptible to apoptosis than myoblasts, which may contribute to depletion of the satellite cell pool. Thus, with age, a subset of myoblasts takes on an altered phenotype, which is marked by high Sca-1 expression. These cells do not participate in muscle regeneration, and instead may contribute to muscle fibrosis in aged muscle.

Nutrition-ethanol (EtOH) interactions during gestation remain unclear primarily due to the lack of appropriate rodent models. In the present report we utilize total enteral nutrition (TEN) to specifically understand the roles of nutrition and caloric intake in EtOH-induced fetal toxicity. Time-impregnated rats were intragastrically fed either control or diets containing EtOH (8–14 g/kg/day) at a recommended caloric intake for pregnant rats or rats 30% undernourished, from gestation day (GD) 6–20. Decreased fetal weight and litter size (P < 0.05) and increased full litter resorptions (33% vs. 0%), were observed in undernourished dams compared to adequately fed rats given the same dose of EtOH, while undernutrition alone did not produce any fetal toxicity. Undernutrition led to impairment of EtOH metabolism, increased blood EtOH concentrations (160%), and decreased maternal hepatic ADH1 mRNA, protein, and activity. Microarray analyses of maternal hepatic gene expression on GD15 revealed that 369 genes were altered by EtOH in the presence of undernutrition, as compared to only 37 genes by EtOH per se (±2-fold, P < 0.05). Hierarchical clustering and gene ontology analysis revealed that stress and external stimulus responses, transcriptional regulation, cellular homeostasis, and protein metabolism were affected uniquely in the EtOH-under-nutrition group, but not by EtOH alone. Microarray data were confirmed using real-time RT-PCR. Undernourished EtOH-fed animals had 2-fold lower IGF-1 mRNA and 10-fold lower serum IGF-1 protein levels compared to undernourished controls (P < 0.0005). Examination of maternal GH signaling via STAT5a and -5b revealed significant reduction in both gene and protein expression produced by both EtOH and undernutrition. However, despite significantly elevated fetal BECs, fetal IGF-1 mRNA and protein were not affected by EtOH or EtOH-undernutrition combinations. Our data suggest that undernutrition potentiates the fetal toxicity of EtOH in part by disrupting maternal GH-IGF-1, signaling thereby decreasing maternal uterine capacity and placental growth.

Chronic ethanol (EtOH) consumption can result in osteopenia. In the current study, we examined the modulation of EtOH-induced bone loss during pregnancy. Nonpregnant and pregnant dams were intragastrically infused either control or EtOH-containing diets throughout gestation (gestation d 5 through 20 or an equivalent period of 15 d) by total enteral nutrition. The effects of EtOH (8.5 to 14 g/kg/d) on tibial bone mineral density (BMD), mineral content (BMC), and bone mineral area were assessed at gestation d 20 via peripheral quantitative computerized tomography. EtOH caused a dose-dependent decrease in BMD and BMC without affecting bone mineral area. Trabecular BMD and BMC were significantly lower in EtOH-treated, nonpregnant dams, compared with pregnant cohorts at the same infused dose of EtOH and urinary ethanol concentrations. Static histomorphometric analysis of tibiae from pregnant rats after EtOH treatment showed decreased osteoblast and osteoid surface, indicating inhibited bone formation, whereas EtOH-treated cycling rats showed higher osteoclast and eroded surface, indicative of increased bone resorption. Circulating osteocalcin and 1,25-dihydroxyvitamin D3 were lower in both EtOH-fed nonpregnant and pregnant rats. Gene expression of osteoclast markers, 70 kDa v-ATPase, and tartrate-resistant acid phosphatase were increased selectively in nonpregnant EtOH-treated rats but not pregnant rats. Moreover, only nonpregnant EtOH-fed rats showed induction in bone marrow receptor activator of nuclear factor-κB ligand mRNA and decreased circulating 17β-estradiol levels. Our data suggest that EtOH-induced bone loss in pregnant rats is mainly due to inhibited bone formation, whereas in nonpregnant rats, the data are consistent with increased osteoclast activation and bone resorption concomitant with decreased estradiol levels.

The polymorphic cytochrome P450 2D6 (CYP2D6) is involved in the metabolism of 30% of the drugs currently prescribed, and is thus clinically relevant. Typical CYP2D6 substrates generally contain a basic nitrogen atom and an aromatic moiety adjacent to the site of metabolism. Recently, we demonstrated the importance of active site residue F120 in substrate binding and catalysis in CYP2D6. On the basis of protein homology models, it is claimed that another active site phenylalanine, F483, may also play an important role in the interaction with the aromatic moiety of CYP2D6 substrates. Experimental data to support this hypothesis, however, is not yet available. In fact, in the only study performed, mutation of F483 to isoleucine or tryptophan did not affect the 1'-hydroxylation of bufuralol at all [Smith G, Modi S, Pillai I, Lian LY, Sutcliffe MJ, Pritchard MP, et al., Determinants of the substrate specificity of human cytochrome P-450 CYP2D6: design and construction of a mutant with testosterone hydroxylase activity. Biochem J 1998;331:783-92]. In the present study, the role of F483 in ligand binding and metabolism by CYP2D6 was examined experimentally using site-directed mutagenesis. Replacement of F483 by alanine resulted in a 30-fold lower V(max) for bufuralol 1'-hydroxylation, while the K(m) was hardly affected. The V(max) for 3,4-methylenedioxy-methylamphetamine O-demethylenation on the other hand decreased only two-fold, whereas the effect on the K(m) was much larger. For dextromethorphan, in addition to dextrorphan (O-demethylation) and 3-methoxymorphinan (N-demethylation), two other metabolites were formed that could not be detected for the wild-type. The substrate 7-methoxy-4-(aminomethyl)-coumarin was not metabolised at all by CYP2D6[F483A], a phenomenon that was reported also for CYP2D6[F120A]. The presented data show that next to F120, residue F483 plays a very important role in the metabolism of typical CYP2D6 substrates. The influence of F483 on metabolism was found to be strongly substrate-dependent.

We have studied the effects of pregnancy on ethanol clearance rates and on blood and urine ethanol concentrations (BECs and UECs) in adult Sprague-Dawley rats infused with ethanol intragastrically. Pregnant rats had greater ethanol clearance following an intragastric or intravenous ethanol bolus (3 or 0.75 g/kg, respectively) relative to non-pregnant rats (p

A comparative metabolism study was performed for bufuralol, dextromethorphan, imipramine, mianserin, sparteine, tamoxifen, haloperidol and two drug candidates (Rec27/0110 and Rec15/2739) on V79 cells genetically engineered to express human cytochrome P450 (CYP) variants 2D6*1, 2D*2, 2D*9 and 2D*17. Unexpectedly, the CYP2D6*17 dependent metabolism profile of haloperidol and Rec27/0110 were found to differ from all other substrates tested. Some of these known standard substrates are frequently applied in marker reactions for CYP2D6 and with these standard substrates, CYP2D6*1 is known to be the most active form. In both cases of haloperidol and Rec27/0110 the variant form CYP2D6*17 had equal or higher activity compared to the CYP2D6*1 form. Results obtained with the V79 cells were confirmed using microsomal preparation of yeast cells expressing the variants CYP2D6*1 and CYP2D6*17 and CYP2D6 inhibitor quinidine. In conclusion, there is no general rule for a variant dependent metabolism profile by cytochrome P450 2D6 indicating that the activity profile of the CYP2D6 alleles may be substrate specific, thus may be reflected in pharmacokinetics consequences for individuals.

The role of the hydrophobic NH(2)-terminal domain of rat CYP2E1 for intracellular targeting and membrane binding was investigated in Saccharomyces cerevisiae as a model system. Several different CYP2E1 variants with deletions and mutations were expressed in yeast, and their intracellular localization and membrane-binding properties were analyzed. We found that an amino acid stretch including the B-helix from glycine 82 to asparagine 95 is responsible for mitochondrial association of CYP2E1 in yeast. Furthermore, we investigated the membrane-binding properties of the variants and concluded that the same region in the B-helix is responsible for membrane interactions of CYP2E1 by electrostatic interactions. A soluble variant of CYP2E1 lacking the first 82 amino acids and containing leucine to aspartate amino acid exchanges at positions 90 and 91, which disrupted the amphipathic nature of the B-helix, was expressed at relatively high levels in the yeast and was found to be catalytically active toward chlorzoxazone in cumene hydroperoxide-supported reactions. We suggest that these amino acid changes at positions 90 and 91 abolish the electrostatic interaction between the negatively charged membrane and the positively charged B-helix, thereby producing a soluble product.

Selegiline was used as a model compound in a project aimed at comparing, evaluating, and integrating different in vitro approaches for the prediction of cytochrome P450 (P450)-catalyzed hepatic drug metabolism in humans (EUROCYP). Metabolic predictions were generated using homology modeling, cDNA-expressed P450 enzymes, human liver microsomes, primary cultured human hepatocytes, and precision-cut human liver slices. All of the in vitro systems correctly indicated the formation of two dealkylated metabolites, desmethylselegiline and methamphetamine. The metabolic instability of selegiline was demonstrated by all of the in vitro systems studied. Estimates of clearance varied from 16 l/h to 223 l/h. With the exception of one approach, all systems underpredicted the in vivo clearance in humans (236 l/h). Despite this, all approaches successfully classified selegiline as a high clearance compound. Homology modeling suggested the participation of CYP2B6 in the demethylation of selegiline and of CYP2D6 in the depropargylation of the drug. Studies with recombinant expressed enzymes and with human hepatic microsomal fraction supported the involvement of CYP2B6 but not of CYP2D6. These techniques also suggested the involvement of CYP1A2, CYP2C8, and CYP2C19 in the biotransformation of selegiline. In vitro, CYP2B6 was the most active form of P450 involved in selegiline metabolism. Metabolism by several enzymes operating in parallel implies a low interaction potential for the drug. None of the techniques alone was able to predict all aspects of the metabolic and kinetic behavior of selegiline in vivo. However, when used as an integrated package, all significant characteristics were predictable.

The 14th International Symposium on Microsomes and Drug Oxidations was held July 22­26, 2002, in Sapporo, Japan. The meeting included 13 plenary sessions, 75 symposia lectures and about 200 poster presentations, and covered a wide variety of topics on general, clinical and toxicologic aspects of drug metabolism. Highlights of the meeting are discussed. (c) 2002 Prous Science. All rights reserved.

Cytochrome P450 1B1 (CYP1B1) is an extrahepatic enzyme of potential importance for the metabolism of estrogen and for metabolic activation of environmental carcinogens. We investigated an Ethiopian population for functional polymorphisms in the CYP1B1 gene using genomic DNA sequencing and detected three novel single nucleotide polymorphisms (SNPs). One of these (4360C-->G in exon 3) is present at a frequency of 7% and causes an Ala443Gly amino acid substitution. In addition, the four described previously missense mutations Arg48Gly, Ala119Ser, Leu432Val, and Asn453Ser were found with frequencies of 51, 50, 53, and 2%, respectively, yielding a total of 32 possible CYP1B1 haplotypes. Allele-specific PCR methods for haplotype analysis were developed and seven different CYP1B1 alleles were found: CYP1B1*1, *2, *3, *4, *5, *6, and *7 with frequencies of 8, 37, 39, 2, 0.7, 6, and 7%, respectively. The functional properties of different forms of CYP1B1, as well as of the Leu432Val + Asn453Ser and Leu432Val + Ala443Gly variants, were evaluated after heterologous expression of the corresponding cDNAs in Saccaromyces cerevisiae. The results revealed that CYP1B1.6 and CYP1B1.7, having the amino acid substitutions Arg48Gly, Ala119Ser, and Leu432Val in common, exhibited altered kinetics with significantly increased apparent K(m) and lowered V(max) values for both the 2- and 4-hydroxylation of 17 beta-estradiol, whereas the other constructs were indistinguishable from the CYP1B1.1 enzyme. The results emphasize the necessity of a complete haplotype analysis of enzyme variants for evaluation of functional consequences in vivo and for analyses of genetic polymorphisms in relation to, for example, cancer incidence.

Porcine CYP2D25, microsomal vitamin D(3) 25-hydroxylase, catalyzes the essential first step in the bioactivation of the prohormone vitamin D(3). Although CYP2D25 shows a high degree of sequence identity with other members of the CYP2D subfamily, such as human CYP2D6, the vitamin D(3) 25-hydroxylase activity is a unique property among CYP2D enzymes. In addition to 25-hydroxylation, CYP2D25 also metabolizes the drug tolterodine. In this study, CYP2D25 was functionally expressed in the Saccharomyces cerevisiae W(R) strain and site-directed mutagenesis was used to study the role of substrate recognition site 3 (SRS-3) for the catalytic specificity of CYP2D25. Five residues in SRS-3 of CYP2D25 were simultaneously mutated to the equivalent residues in CYP2D6, an enzyme not active in 25-hydroxylation. Western blot analysis of microsomes from transformed yeast cells showed that both the wild-type and mutant CYP2D25 were expressed at comparable levels. The 25-hydroxylase activity of recombinant mutant CYP2D25 was completely lost whereas the activity toward tolterodine remained virtually unaffected. The results implicate that residues in SRS-3 of CYP2D25 are important determinants for its function in vitamin D(3) metabolism.

Debrisoquine metabolism among Tanzanians has been found to be slower than expected from the CYP2D6 genotype. In order to evaluate any genetic explanation, the coding sequence and intron-exon boundaries of the CYP2D6 gene from three Black Tanzanian volunteers with a CYP2D6*1/*1 or CYP2D6*2/*2 genotype and debrisoquine metabolic ratios (MRs) > 1 were fully sequenced to screen for new mutations. Two functional mutations, G1747 to A (causing V136I) and G3271 to A (causing V338M), were identified in the CYP2D6*2/*2 sample. Thirty-six subjects (34%) out of a total 106 subjects were heterozygous and three subjects (3%) were homozygous for the allele, yielding an allele frequency of 20%. The CYP2D6*29 allele, having also the mutations of the CYP2D6*2 allele, was subsequently expressed in yeast and mammalian COS-1 cells. No differences were seen with respect to the affinity (Km) or maximal velocity (Vmax) of the CYP2D6 substrate bufuralol between the wild-type and mutant when expression was carried out in yeast cells. By contrast, the 1'-hydroxybufuralol catalytic activity of the mutant expressed in COS-1 cells was only 26% of the wild-type (P < 0.01; Mann-Whitney U-test) and its debrisoquine hydroxylation activity was 63% of that of CYP2D6.1. The single mutants V136I and V338M had reduced capacity for bufuralol hydroxylation, but the effect was even stronger when both mutations were present together as in CYP2D6.29. Analysis of the distribution of CYP2D6*29 in subjects phenotyped for debrisoquine revealed that this allele significantly causes a reduction in the rate of debrisoquine hydroxylation in vivo. The results indicate the common existence in Tanzanians of a variant CYP2D6 form with different substrate specificity as compared to the wild-type form of the enzyme causing reduced capacity for debrisoquine metabolism.

A novel cytochrome P450, CYP4F8, was recently cloned from human seminal vesicles. CYP4F8 was expressed in yeast. Recombinant CYP4F8 oxygenated arachidonic acid to (18R)-hydroxyarachidonate, whereas prostaglandin (PG) D(2), PGE(1), PGE(2), PGF(2alpha), and leukotriene B(4) appeared to be poor substrates. Three stable PGH(2) analogues, 9,11-epoxymethano-PGH(2) (U-44069), 11, 9-epoxymethano-PGH(2) (U-46619), and 9,11-diazo-15-deoxy-PGH(2) (U-51605) were rapidly metabolized by omega2- and omega3-hydroxylation. U-44069 was oxygenated with a V(max) of approximately 260 pmol min(-)(1) pmol P450(-1) and a K(m) of approximately 7 micrometer. PGH(2) decomposes mainly to PGE(2) in buffer and to PGF(2alpha) by reduction with SnCl(2). CYP4F8 metabolized PGH(2) to 19-hydroxy-PGH(2), which decomposed to 19-hydroxy-PGE(2) in buffer and could be reduced to 19-hydroxy-PGF(2alpha) with SnCl(2). 18-Hydroxy metabolites were also formed (approximately 17%). PGH(1) was metabolized to 19- and 18-hydroxy-PGH(1) in the same way. Microsomes of human seminal vesicles oxygenated arachidonate, U-44069, U-46619, U-51605, and PGH(2), similar to CYP4F8. (19R)-Hydroxy-PGE(1) and (19R)-hydroxy-PGE(2) are the main prostaglandins of human seminal fluid. We propose that they are formed by CYP4F8-catalyzed omega2-hydroxylation of PGH(1) and PGH(2) in the seminal vesicles and isomerization to (19R)-hydroxy-PGE by PGE synthase. CYP4F8 is the first described hydroxylase with specificity and catalytic competence for prostaglandin endoperoxides.