Show simple item record

dc.contributor.authorGonzalez Covarrubias, Mirelle Vanessa
dc.date.accessioned2016-03-21T20:43:05Z
dc.date.available2016-03-21T20:43:05Z
dc.date.issued2008
dc.identifier.isbn9780549765578
dc.identifier.other304387058
dc.identifier.urihttp://hdl.handle.net/10477/43444
dc.description.abstractThis research utilized a human liver bank consisting of 96-paired DNA-RNA-tissue (blacks n = 32 and whites n = 64) samples processed at St. Jude Children's Research Hospital, and were provided by the Liver Tissue Procurement and Distribution System (NIH Contract #N01-DK-9-2310), and by the Cooperative Human Tissue Network. Chapter 1 investigated the relative contributions of CBR and NAD(P)H:quinone oxidoreductase (NQO1) activities to the total quinone-reducing capacity of human liver. We utilized the quinone substrate menadione and the specific NQO1 inhibitor, dicumarol to discriminate between CBR and NQO1 enzymatic activities in liver cytosolic fractions. CBR activity accounted for most of the quinone reducing capacity of human liver in black (85%) and white (93%) donors. CBR activity varied widely in both populations (whites, > 28-fold and blacks, 5-fold). We detected a 2-fold higher NQO1 activity in samples from blacks compared to whites. There was a significant genotype-phenotype correlation for the NQO1*2 polymorphism and NQO1 cytosolic activity in samples from white donors. Samples with NQO1*1/*1 genotype showed 2.6-fold higher NQO1 activity than samples with NQO1*1/*2 plus NQO1*2/*2 genotypes. This study documented the relative contributions of hepatic CBR and NQO1 activities to the quinone reducing capacity of human liver. We pinpointed interethnic differences in NQO1 activity and confirmed the impact of the NQO1*2 polymorphism in human liver that has been previously reported for other tissues. To determine genetic determinants of variable CBR activity we screened CBR1 full length cDNA samples and performed database search analyses. Chapter 2 described the functional characterization of the CBR1 V88I polymorphism. We cloned and expressed CBR1 V88 and CBR1 I88 proteins to perform kinetic experiments. The CBR1 V88 variant showed 1.7-fold higher V max values for daunorubicin and PGE2 compared to the CBR1 I88 isoform. Consistently, CBR1 V88 synthesized 47% more daunorubicinol than CBR1 I88. Isothermal titration calorimetry analyses together with molecular modeling simulations showed that CBR1 V88I results in CBR1 isoforms with different binding affinities for the cofactor NADPH (K d CBR1 V88 = 6.3 ± 0.6 μM vs. K d CBR1 I88 = 3.8 ± 0.5 μM). These studies characterize the first functional genetic determinant of CBR1 activity towards relevant physiological and pharmacological substrates. CBR1 reduces the anthracyclines doxorubicin and daunorubicin into doxorubicinol and daunorubicinol. These alcohol metabolites advace the development of anthracycline-related cardiotoxicity. Hence, the pharmacological inhibition of CBR1 activity has been proposed as a promising strategy to minimize the incidence of anthracycline-related cardiotoxicity. The semisynthetic flavonoid, monoHER has been extensively investigated as a potential cardioprotectant against anthracycline-related cardiotoxicity. In Chapter 3 , we investigated the inhibitory properties (IC 50 ) of monoHER and the structurally related flavonoids, quercetin and triHER, on polymorphic CBR1 V88I. MonoHER, triHER, and quercetin showed significantly lower IC 50 values for CBR1 I88 compared to those for CBR1 V88 in the presence of the substrates menadione, daunorubicin and doxorubicin. Inhibition of polymorphic CBR1 by monoHER showed 3-fold higher IC 50 values for daunorubicin compared to monoHER IC 50 values for doxorubicin. MonoHER inhibited CBR1 competitively in the presence of daunorubicin (Ki = 45 ± 18 μM) and uncompetitively in the presence of menadione (Ki = 45 ± 18 μM). These results showed that the pharmacological inhibition of CBR1 activity would be dictated by the type of anthracycline substrate and by CBR1 V88I genotype status. CBR-mediated metabolism of anthracyclines displays a wide interindividual variability. It is possible that variations in CBR1 expression contribute to the unpredictable pharmacokinetics and pharmacodynamics of these anticancer drugs. Chapter 4 documented the extent of interethnic and interindividual variations of hepatic CBR1 expression at the mRNA, protein, and CBR activity levels in 97-paired DNA-RNA-liver tissue samples from black and white donors. We determined the degree of concordance between self-identified ethnicity and distinctive geographical ancestries using a panel of 176 ancestry informative markers (AIM). Stratification by ethnicity or AIM-determined geographical ancestry did not modify the average values of hepatic CBR1 mRNA, CBR1 protein or CBR activity. Therefore, in this group of samples self-reported ethnicity appears to be an accurate indicator of geographical ancestry. CBR1 mRNA expression and protein levels did not differ between blacks and whites. CBR activity, expressed as the synthesis of doxorubicinol, varied widely among individuals (CBR activity blacks: 0.87-8.7 nmol doxol/min.mg and CBR activity whites: 0.4-8.4 nmol doxol/min.mg; p = 0.610). There was a negative correlation between age and doxorubicinol synthesis in samples from whites (r p = -0.434, p = 0.006), but not in samples from blacks. This study suggests that variable CBR1 expression may impact on the pharmacokinetics of the anticancer doxorubicin. In chapter 5 , we sought to identify genetic determinants of variable CBR1 expression. We investigated the presence of single nucleotide polymorphisms on CBR1 (e.g. CBR1 1096G>A, CBR1 V88I, and CBR1 S131P) in DNA variation panels and in DNA samples from black and white donors. (Abstract shortened by UMI.)
dc.languageEnglish
dc.subjectHealth and environmental sciences
dc.subjectCarbonyl reductase
dc.subjectLiver
dc.subjectAnthracyclines
dc.subjectSingle nucleotide polymorphisms
dc.subjectDrug metabolism
dc.titlePharmacogenetics of human carbonyl reductase 1 (CBR1) in liver from black and white donors
dc.typeDissertation/Thesis


Files in this item

Thumbnail

This item appears in the following Collection(s)

Show simple item record