Sevasti B. Koukouritaki

Human hepatic flavin-containing monooxygenases 1 (FMO1) and 3 (FMO3) developmental expression

The flavin-containing monooxygenases (FMOs) are important for the metabolism of numerous therapeutics and toxicants. Six mammalian FMO genes (FMO1–6) have been identified, each exhibiting developmental and tissue- and species-specific expression patterns. Previous studies demonstrated that human hepatic FMO1 is restricted to the fetus whereas FMO3 is the major adult isoform. These studies failed to describe temporal expression patterns, the precise timing of the FMO1/FMO3 switch, or potential control mechanisms. To address these questions, FMO1 and FMO3 were quantified in microsomal fractions from 240 human liver samples representing ages from 8 wk gestation to 18 y using Western blotting. FMO1 expression was highest in the embryo (8–15 wk gestation; 7.8 ± 5.3 pmol/mg protein). Low levels of FMO3 expression also were detectable in the embryo, but not in the fetus. FMO1 suppression occurred within 3 d postpartum in a process tightly coupled to birth, but not gestational age. The onset of FMO3 expression was highly variable, with most individuals failing to express this isoform during the neonatal period. FMO3 was detectable in most individuals by 1-2 y of age and was expressed at intermediate levels until 11 y (12.7 ± 8.0 pmol/mg protein). These data suggest that birth is necessary, but not sufficient for the onset of FMO3 expression. A gender-independent increase in FMO3 expression was observed from 11 to 18 y of age (26.9 ± 8.6 pmol/mg protein). Finally, 2- to 20-fold interindividual variation in FMO1 and FMO3 protein levels were observed, depending on the age bracket.

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.

DEVELOPMENTAL EXPRESSION OF ARYL, ESTROGEN AND HYDROXYSTEROID SULFOTRANSFERASES IN PRE- AND POSTNATAL HUMAN LIVER

Aryl- (SULT1A1), estrogen- (SULT1E1), and hydroxysteroid- (SULT2A1) sulfotransferases (SULTs) are active determinants of xenobiotic detoxication and hormone metabolism in the adult human liver. To investigate the role of these conjugating enzymes in the developing human liver, the ontogeny of immunoreactive SULT1A1, SULT1E1, and SULT2A1 expression was characterized in a series of 235 pre- and postnatal human liver cytosols ranging in age from early gestation to a postnatal age of 18 years. Interindividual variability in expression levels was apparent for all three SULTs in pre- and postnatal liver samples. Expression of the three SULTs displayed distinctly different developmental profiles. Semiquantitative Western blot analyses indicated that SULT1A1 and SULT2A1 immunoreactive protein levels were readily detectable in the majority of developmental human liver cytosols throughout the prenatal period. Whereas SULT1A1 expression did not differ significantly among the various developmental stages, SULT2A1 expression increased during the third trimester of gestation and continued to increase during postnatal life. By contrast, SULT1E1, a cardinal estrogen-inactivating enzyme, achieved the highest levels of expression during the earliest periods of gestation in prenatal male livers, indicating a requisite role for estrogen inactivation in the developing male. The present analysis suggests that divergent regulatory mechanisms are responsible for the differential patterns of hepatic SULT1A1, SULT1E1, and SULT2A1 immunoreactive protein levels that occur during pre- and postnatal human development, and implicates a major role for sulfotransferase expression in the developing fetus.

Identification and functional analysis of common human flavin-containing monooxygenase 3 genetic variants

Flavin-containing monooxygenases (FMOs) are important for the disposition of many therapeutics, environmental toxicants, and nutrients. FMO3, the major adult hepatic FMO enzyme, exhibits significant interindividual variation. Eighteen FMO3 single-nucleotide polymorphism (SNP) frequencies were determined in 202 Hispanics (Mexican descent), 201 African Americans, and 200 non-Latino whites. Using expressed recombinant enzyme with methimazole, trimethylamine, sulindac, and ethylenethiourea, the novel structural variants FMO3 E24D and K416N were shown to cause modest changes in catalytic efficiency, whereas a third novel variant, FMO3 N61K, was essentially devoid of activity. The latter variant was present at an allelic frequency of 5.2% in non-Latino whites and 3.5% in African Americans, but it was absent in Hispanics. Inferring haplotypes using PHASE, version 2.1, the greatest haplotype diversity was observed in African Americans followed by non-Latino whites and Hispanics. Haplotype 2A and 2B, consisting of a hypermorphic promoter SNP cluster (-2650C>G, -2543T>A, and -2177G>C) in linkage with synonymous structural variants was inferred at a frequency of 27% in the Hispanic population, but only 5% in non-Latino whites and African Americans. This same promoter SNP cluster in linkage with one or more hypomorphic structural variant also was inferred in multiple haplotypes at a total frequency of 5.6% in the African-American study group but less than 1% in the other two groups. The sum frequencies of the hypomorphic haplotypes H3 [15,167G>A (E158K)], H5B [-2650C>G, 15,167G>A (E158K), 21,375C>T (N285N), 21,443A>G (E308G)], and H6 [15,167G>A (E158K), 21,375C>T (N285N)] was 28% in Hispanics, 23% in non-Latino whites, and 24% in African Americans.

Flavin-containing monooxygenase genetic polymorphism: impact on chemical metabolism and drug development.

The flavin-containing monooxygenases (FMOs) metabolize a broad range of therapeutics. Consisting of five gene products in humans (FMO1-5), the different FMO family members exhibit pronounced tissue- and temporal-specific expression patterns. Substantial interindividual differences are also observed, and the inability to modulate with exogenous agents is consistent with an important role for genetic variation. Several rare FMO3 alleles causative for trimethylaminuria have been well characterized. However, the identification and characterization of functional FMO1-5 polymorphisms has been more recent. Although none of these polymorphisms has been associated with an adverse drug reaction, the continued broadening of our therapeutic armamentarium makes such an event likely in the future. Furthermore, at least one example has been reported for a direct association between FMO3 polymorphism and therapeutic efficacy. Thus, it is anticipated that knowledge regarding functionally-relevant FMO genetic variability will become increasingly important for making drug development and patient therapeutic choices.

Platelet-targeted gene therapy with human factor VIII establishes haemostasis in dogs with haemophilia A

It is essential to improve therapies for controlling excessive bleeding in patients with haemorrhagic disorders. As activated blood platelets mediate the primary response to vascular injury, we hypothesize that storage of coagulation Factor VIII within platelets may provide a locally inducible treatment to maintain haemostasis for haemophilia A. Here we show that haematopoietic stem cell gene therapy can prevent the occurrence of severe bleeding episodes in dogs with haemophilia A for at least 2.5 years after transplantation. We employ a clinically relevant strategy based on a lentiviral vector encoding the ITGA2B gene promoter, which drives platelet-specific expression of human FVIII permitting storage and release of FVIII from activated platelets. One animal receives a hybrid molecule of FVIII fused to the von Willebrand Factor propeptide-D2 domain that traffics FVIII more effectively into α-granules. The absence of inhibitory antibodies to platelet-derived FVIII indicates that this approach may have benefit in patients who reject FVIII replacement therapies. Thus, platelet FVIII may provide effective long-term control of bleeding in patients with haemophilia A.

Enzyme-Mediated Protein Haptenation of Dapsone and Sulfamethoxazole in Human Keratinocytes: II. Expression and Role of Flavin-Containing Monooxygenases and Peroxidases

Arylamine compounds, such as sulfamethoxazole (SMX) and dapsone (DDS), are metabolized in epidermal keratinocytes to arylhydroxylamine metabolites that auto-oxidize to arylnitroso derivatives, which in turn bind to cellular proteins and can act as antigens/immunogens. Previous studies have demonstrated that neither cytochromes P450 nor cyclooxygenases mediate this bioactivation in normal human epidermal keratinocytes (NHEKs). In this investigation, we demonstrated that methimazole (MMZ), a prototypical substrate of the flavin-containing monooxygenases (FMOs), attenuated the protein haptenation observed in NHEKs exposed to SMX or DDS. In addition, recombinant FMO1 and FMO3 were able to bioactivate both SMX and DDS, resulting in covalent adduct formation. Western blot analysis confirmed the presence of FMO3 in NHEKs, whereas FMO1 was not detectable. In addition to MMZ, 4-aminobenzoic acid hydrazide (ABH) also attenuated SMX- and DDS-dependent protein haptenation in NHEKs. ABH did not alter the bioactivation of these drugs by recombinant FMO3, suggesting its inhibitory effect in NHEKs was due to its known ability to inhibit peroxidases. Studies confirmed the presence of peroxidase activity in NHEKs; however, immunoblot analysis and reverse transcription-polymerase chain reaction indicated that myeloperoxidase, lactoperoxidase, and thyroid peroxidase were absent. Thus, our results suggest an important role for FMO3 and yet-to-be identified peroxidases in the bioactivation of sulfonamides in NHEKs.

Platelet gene therapy improves hemostatic function for integrin αIIbβ3-deficient dogs

Activated blood platelets mediate the primary response to vascular injury. Although molecular abnormalities of platelet proteins occur infrequently, taken collectively, an inherited platelet defect accounts for a bleeding diathesis in ≈1:20,000 individuals. One rare example of a platelet disorder, Glanzmann thrombasthenia (GT), is characterized by life-long morbidity and mortality due to molecular abnormalities in a major platelet adhesion receptor, integrin αIIbβ3. Transfusion therapy is frequently inadequate because patients often generate antibodies to αIIbβ3, leading to immune-mediated destruction of healthy platelets. In the most severe cases allogeneic bone marrow transplantation has been used, yet because of the risk of the procedure it has been limited to few patients. Thus, hematopoietic stem cell gene transfer was explored as a strategy to improve platelet function within a canine model for GT. Bleeding complications necessitated the use of a mild pretransplant conditioning regimen; therefore, in vivo drug selection was used to improve engraftment of autologously transplanted cells. Approximately 5,000 αIIbβ3 receptors formed on 10% of platelets. These modest levels allowed platelets to adhere to αIIbβ3’s major ligand (fibrinogen), form aggregates, and mediate retraction of a fibrin clot. Remarkably, improved hemostatic function was evident, with ≤135-fold reduced blood loss, and improved buccal bleeding times decreased to 4 min for up to 5 y after transplant. One of four transplanted dogs developed a significant antibody response to αIIbβ3 that was attenuated effectively with transient immune suppression. These results indicate that gene therapy could become a practical approach for treating inherited platelet defects.

High-level transgene expression in induced pluripotent stem cell–derived megakaryocytes: correction of Glanzmann thrombasthenia

Megakaryocyte-specific transgene expression in patient-derived induced pluripotent stem cells (iPSCs) offers a new approach to study and potentially treat disorders affecting megakaryocytes and platelets. By using a Gp1ba promoter, we developed a strategy for achieving a high level of protein expression in human megakaryocytes. The feasibility of this approach was demonstrated in iPSCs derived from two patients with Glanzmann thrombasthenia (GT), an inherited platelet disorder caused by mutations in integrin αIIbβ3. Hemizygous insertion of Gp1ba promoter-driven human αIIb complementary DNA into the AAVS1 locus of iPSCs led to high αIIb messenger RNA and protein expression and correction of surface αIIbβ3 in megakaryocytes. Agonist stimulation of these cells displayed recovery of integrin αIIbβ3 activation. Our findings demonstrate a novel approach to studying human megakaryocyte biology as well as functional correction of the GT defect, offering a potential therapeutic strategy for patients with diseases that affect platelet function.

Enhanced expression of the cytoskeleton-associated proteins paxillin and focal adhesion kinase in glomerular immune injury.

In a rat model of glomerular immune injury induced by antibody against the glomerular basement membrane (GBM), we assessed changes in the levels and in the extent of tyrosine phosphorylation of two cytoskeleton-associated proteins, focal adhesion kinase (FAK) and paxillin. Glomeruli were isolated 2, 7, and 14 days after the administration of a rabbit anti-rat GBM antibody that induced proliferative nephritis and proteinuria. FAK and paxillin levels in glomerular protein lysates were assessed by immunoprecipitation followed by Western blot analysis. Changes in the tyrosine phosphorylation of immunoprecipitated paxillin and FAK were assessed by Western blot analysis with antiphosphotyrosine antibodies. Glomerular levels of FAK and paxillin were increased in nephritic glomeruli as compared with non-nephritic controls at all time points. There was a discordant increase in the tyrosine phosphorylation levels of paxillin and FAK; the increase in the tyrosine phosphorylation of FAK was sustained and peaked on day 7 of immune injury, whereas that of paxillin was short-lived and peaked on day 2 of injury. We propose that these changes in FAK and paxillin expression and tyrosine phosphorylation reflect interactions between glomerular cells and accumulating extracellular matrix proteins in the course of immune injury, or they constitute parts of wider signaling events within the nephritic glomerulus that involve the cytoskeleton.