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Deanna L Kroetz

Publications and source records attributed to Deanna L Kroetz.

11 recordsLinked to original sources
PubMed2004

Functional implications of genetic polymorphisms in the multidrug resistance gene MDR1 (ABCB1).

The multidrug resistance (MDR1) gene product P-glycoprotein is a membrane protein that functions as an ATP-dependent efflux pump, transporting exogenous and endogenous substrates from the inside of cells to the outside. Physiological expression of P-glycoprotein in tissues with excretory or protective function is a major determinant of drug disposition and provides a cellular defense mechanism against potentially harmful compounds. Therefore, P-glycoprotein has significant impact on therapeutic efficacy and toxicity as it plays a key role in absorption of oral medications from the intestinal tract, excretion into bile and urine, and distribution into protected tissues such as the brain and testes. There is increasing interest in the possible role of genetic variation in MDR1 in drug therapy. Numerous genetic polymorphisms in MDR1 have been described, some of which have been shown to determine P-glycoprotein expression levels and substrate transport. Furthermore, some of these polymorphisms have an impact on pharmacokinetic and pharmacodynamic profiles of drug substrates and directly influence outcome and prognosis of certain diseases. This review will focus on the impact of genetic variation in MDR1 on expression and function of P-glycoprotein and the implications of this variation for drug therapy and disease risk.

Genes, MDR↗
PubMed2003

Vascular localization of soluble epoxide hydrolase in the human kidney.

Epoxyeicosatrienoic acids are cytochrome P-450 metabolites of arachidonic acid with multiple biological functions, including the regulation of vascular tone, renal tubular transport, cellular proliferation, and inflammation. Epoxyeicosatrienoic acids are converted by soluble epoxide hydrolase into the corresponding dihydroxyeicosatrienoic acids, and epoxyeicosatrienoic acid hydration is regarded as one mechanism whereby their biological effects are eliminated. Previous animal studies indicate that soluble epoxide hydrolase plays an important role in the regulation of renal eicosanoid levels and systemic blood pressure. To begin to elucidate the mechanism of these effects, we determined the cellular localization of soluble epoxide hydrolase in human kidney by examining biopsies taken from patients with a variety of non-end-stage renal diseases, as well as those without known renal disease. Immunohistochemical staining of acetone-fixed kidney biopsy samples revealed that soluble epoxide hydrolase was preferentially expressed in the renal vasculature with relatively low levels in the surrounding tubules. Expression of soluble epoxide hydrolase was evident in renal arteries of varying diameter and was localized mostly in the smooth muscle layers of the arterial wall. Western blot analysis and functional assays confirmed the expression of soluble epoxide hydrolase in the human kidney. There were no obvious differences in soluble epoxide hydrolase expression between normal and diseased human kidney tissue in the samples examined. Our results indicate that soluble epoxide hydrolase is present in the human kidney, being preferentially expressed in the renal vasculature, and support an essential role for this enzyme in renal hemodynamic regulation and its potential utility as a target for therapeutic intervention.

Arterioles↗
PubMed2003

Catalytic activity and isoform-specific inhibition of rat cytochrome p450 4F enzymes.

Arachidonic acid is omega-hydroxylated to 20-hydroxyeicosatetraenoic acid (20-HETE), which has effects on vasoactivity and renal tubular transport and has been implicated in the regulation of blood pressure. Cytochrome p450 (p450) 4A isoforms are generally considered the major arachidonic acid omega-hydroxylases; however, little is known about the role of rat CYP4F isoforms in 20-HETE formation. The rat CYP4F isoforms, CYP4F1, CYP4F4, CYP4F5, and CYP4F6, were heterologously expressed in Escherichia coli, and their substrate specificity in fatty acid metabolism was characterized. Substrate-binding assays indicated that leukotriene B(4) (LTB(4)) and arachidonic acid bound CYP4F1 and CYP4F4 in a type-I manner with a K(s) of 25 to 59 microM, and lauric acid bound CYP4F4 poorly. Reconstituted CYP4F1 and CYP4F4 catalyzed the omega-hydroxylation of LTB(4) with a K(m) of 24 and 31 microM, respectively, and CYP4F5 had minor activity in LTB(4) metabolism. Importantly, CYP4F1 and CYP4F4 catalyzed the omega-hydroxylation of arachidonic acid with an apparent k(cat) of 9 and 11 min(-1), respectively. Lauric acid was a poor substrate for all of the CYP4F isoforms, and CYP4F6 had no detectable fatty acid omega-hydroxylase activity. The p450 omega-hydroxylase inhibitors 17-octadecynoic acid, 10-undecynyl sulfate, and N-methylsulfonyl-12,12-dibromododec-11-enamide showed isoform-specific inhibition of CYP4F1- and CYP4F4-catalyzed omega-hydroxylation of arachidonic acid and potency differences between the CYP4A and CYP4F isoforms. These data support a significant role for CYP4F1 and CYP4F4 in the formation of 20-HETE and identify p450 inhibitors that can be used to understand the relative contribution of the CYP4A and CYP4F isoforms to renal 20-HETE formation.

Animals↗
PubMed2003

Natural variation in human membrane transporter genes reveals evolutionary and functional constraints.

Membrane transporters maintain cellular and organismal homeostasis by importing nutrients and exporting toxic compounds. Transporters also play a crucial role in drug response, serving as drug targets and setting drug levels. As part of a pharmacogenetics project, we screened exons and flanking intronic regions for variation in a set of 24 membrane transporter genes (96 kb; 57% coding) in 247 DNA samples from ethnically diverse populations. We identified 680 single nucleotide polymorphisms (SNPs), of which 175 were synonymous and 155 caused amino acid changes, and 29 small insertions and deletions. Amino acid diversity (pi(NS)) in transmembrane domains (TMDs) was significantly lower than in loop domains, suggesting that TMDs have special functional constraints. This difference was especially striking in the ATP-binding cassette superfamily and did not parallel evolutionary conservation: there was little variation in the TMDs, even in evolutionarily unconserved residues. We used allele frequency distribution to evaluate different scoring systems (Grantham, blosum62, SIFT, and evolutionarily conservedevolutionarily unconserved) for their ability to predict which SNPs affect function. Our underlying assumption was that alleles that are functionally deleterious will be selected against and thus under represented at high frequencies and over represented at low frequencies. We found that evolutionary conservation of orthologous sequences, as assessed by evolutionarily conservedevolutionarily unconserved and SIFT, was the best predictor of allele frequency distribution and hence of function. European Americans had an excess of high frequency alleles in comparison to African Americans, consistent with a historic bottleneck. In addition, African Americans exhibited a much higher frequency of population specific medium-frequency alleles than did European Americans.

DNA↗
PubMed2003

No effect of MDR1 C3435T variant on loperamide disposition and central nervous system effects.

BACKGROUND: The MDR1 gene encodes the efflux transporter P-glycoprotein, which is highly expressed in the small intestine and in the blood-brain barrier. A major function of P-glycoprotein is to limit the absorption and central nervous system exposure of numerous xenobiotics. A genetic polymorphism in the MDR1 gene (C3435T) has been associated with changes in the intestinal expression level and function of P-glycoprotein. The aim of this study was to investigate the effect of this polymorphism on disposition and brain entry of the P-glycoprotein substrate loperamide. METHODS: Healthy white volunteers were genotyped for the MDR1 C3435T polymorphism, and a 16-mg oral dose of loperamide was administered to 8 subjects with the 3435TT genotype and 8 subjects with the 3435CC genotype. Plasma levels of loperamide were determined by liquid chromatography-tandem mass spectrometry. Loperamide-induced respiratory depression was detected as the ventilatory response to carbon dioxide and was used as a measure of central nervous system side effects. RESULTS: We found no significant difference in loperamide pharmacokinetics between individuals homozygous for the C and the T alleles in position 3435 of MDR1, as follows: peak plasma drug concentration, 3164 +/- 1053 pg/mL and 3021 +/- 984 pg/mL; area under the concentration-time curve from 0 to 8 hours, 14414 +/- 4756 pg. h/mL and 14923 +/- 6466 pg. h/mL; and time to peak plasma drug concentration, 3.9 +/- 1.4 hours and 3.9 +/- 2.6 hours for the MDR1 3435CC and 3435TT genotypes, respectively (P >.05, for all parameters). Hypercapnic ventilatory response changed only minimally after ingestion of loperamide (the coefficient of variation during the 0- to 8-hour period was 21% +/- 14% for the sample population), and there was no MDR1 3435 genotype-related effect on respiratory response. Carriers of the 2 major MDR1 haplotypes, MDR1*1 and MDR1*13, did not differ in their response to loperamide. CONCLUSION: There was no association between the MDR1 C3435T variation and plasma levels or central nervous system effects of the P-glycoprotein substrate loperamide in a white study population. The MDR1 haplotype structure was quite variable and supports the use of haplotypes instead of single nucleotide polymorphisms in determining clinical consequences of genetic variation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2003

Sequence diversity and haplotype structure in the human ABCB1 (MDR1, multidrug resistance transporter) gene.

OBJECTIVES: There is increasing evidence that polymorphism of the ABCB1 (MDR1) gene contributes to interindividual variability in bioavailability and tissue distribution of P-glycoprotein substrates. The aim of the present study was to (1) identify and describe novel variants in the ABCB1 gene, (2) understand the extent of variation in ABCB1 at the population level, (3) analyze how variation in ABCB1 is structured in haplotypes, and (4) functionally characterize the effect of the most common amino acid change in P-glycoprotein. METHODS AND RESULTS: Forty-eight variant sites, including 30 novel variants and 13 coding for amino acid changes, were identified in a collection of 247 ethnically diverse DNA samples. These variants comprised 64 statistically inferred haplotypes, 33 of which accounted for 92% of chromosomes analyzed. The two most common haplotypes, ABCB1*1 and ABCB1*13, differed at six sites (three intronic, two synonymous, and one non-synonymous) and were present in 36% of all chromosomes. Significant population substructure was detected at both the nucleotide and haplotype level. Linkage disequilibrium was significant across the entire ABCB1 gene, especially between the variant sites found in ABCB1*13, and recombination was inferred. The Ala893Ser change found in the common ABCB1*13 haplotype did not affect P-glycoprotein function. CONCLUSION: This study represents a comprehensive analysis of ABCB1 nucleotide diversity and haplotype structure in different populations and illustrates the importance of haplotype considerations in characterizing the functional consequences of ABCB1 polymorphisms.

Base Sequence↗
PubMed2002

Covalent attachment of the heme prosthetic group in the CYP4F cytochrome P450 family.

We demonstrated earlier that the heme in cytochrome P450 enzymes of the CYP4A family is covalently attached to the protein through an I-helix glutamic acid residue [Hoch, U., and Ortiz de Montellano, P. R. (2001) J. Biol. Chem. 276, 11339-11346]. As the critical glutamic acid residue is conserved in many members of the CYP4F class of cytochrome P450 enzymes, we investigated covalent heme binding in this family of enzymes. Chromatographic analysis indicates that the heme is covalently bound in CYP4F1 and CYP4F4, which have the required glutamic acid residue, but not in CYP4F5 and CYP4F6, which do not. Catalytic turnover of CYP4F4 with NADPH-cytochrome P450 reductase shows that the heme is covalently bound through an autocatalytic process. Analysis of the prosthetic group in the CYP4F5 G330E mutant, into which the glutamic acid has been reintroduced, shows that the heme is partially covalently bound and partially converted to noncovalently bound 5-hydroxymethylheme. The modified heme presumably arises by trapping of a 5-methyl carbocation intermediate by a water molecule. CYP4F proteins thus autocatalytically bind their heme groups covalently in a process that requires a glutamic acid both to generate a reactive (cationic) form of the heme methyl and to trap it to give the ester bond.

Amino Acid Sequence↗
PubMed2002

Cytochrome P450 pathways of arachidonic acid metabolism.

Cytochrome P450s metabolize arachidonic acid to hydroxyeicosatetraenoic acids and epoxyeicosatrienoic acids. These eicosanoids are formed in a tissue and cell-specific manner and have numerous biological functions. Of major interest are the opposing actions of hydroxyeicosatetraenoic and epoxyeicosatrienoic acids within the vasculature. Regio- and stereoisomeric epoxyeicosatrienoic acids have potent vasodilatory properties while 20-hydroxyeicosatetraenoic acid is a potent vasoconstrictor. Both effects are mediated through actions on large-conductance Ca2+-activated K+ channels. Cytochrome P450-derived eicosanoids are also important in the regulation of ion transport, and have recently been shown to influence a number of fundamental biological processes including cellular proliferation, apoptosis, inflammation, and hemostasis. The formation of these functionally relevant eicosanoids is tightly controlled by the expression and activity of the cytochrome P450 epoxygenases and hydroxylases. In addition, soluble epoxide hydrolase catalyzes the hydrolysis of epoxyeicosatrienoic acids to dihydroxyeicosatrienoic acids, and the activity of this enzyme is a critical determinant of tissue epoxyeicosatrienoic and dihydroxyeicosatrienoic acid levels. The intracellular balance between epoxyeicosatrienoic, dihydroxyeicosatrienoic and hydroxyeicosatetraenoic acids influences the biological response to these eicosanoids and alterations in their levels have recently been associated with certain pathological conditions. The involvement of the cytochrome P450-derived eicosanoids in a wide array of biological functions and the observation that levels are altered in pathological conditions suggest that the enzymes involved in the formation and degradation of these fatty acids may be novel therapeutic targets.

Animals↗
PubMed2002

Antihypertensive effect of mechanism-based inhibition of renal arachidonic acid omega-hydroxylase activity.

The cytochrome P-450 eicosanoid 20-hydroxyeicosatetraenoic acid (20-HETE) is a potent vasoconstrictor that is implicated in the regulation of blood pressure. The identification of selective inhibitors of renal 20-HETE formation for use in vivo would facilitate studies to determine the systemic effects of this eicosanoid. We characterized the acetylenic fatty acid sodium 10-undecynyl sulfate (10-SUYS) as a potent and selective mechanism-based inhibitor of renal 20-HETE formation. A single dose of 10-SUYS caused an acute reduction in mean arterial blood pressure in 8-wk-old spontaneously hypertensive rats. The decrease in mean arterial pressure was maximal 6 h after 10-SUYS treatment (17.9 +/- 3.2 mmHg; P < 0.05), and blood pressure returned to baseline levels within 24 h after treatment. Treatment with 10-SUYS was associated with a decrease in urinary 20-HETE formation in vivo and attenuation of the vasoconstrictor response of renal interlobar arteries to ANG II in vitro. These results provide further evidence that 20-HETE plays an important role in the regulation of blood pressure in the spontaneously hypertensive rat.

Animals↗
PubMed2002

Candidate gene approach for pharmacogenetic studies.

Genetic diversity in the form of single nucleotide DNA polymorphisms (SNPs) contributes to variable disease susceptibility and drug response. The candidate gene approach has been widely used to identify the genetic basis for pharmacogenetic traits and becomes increasingly more powerful with the recent advances in genomic technologies. High-throughput sequencing and SNP genotyping technologies allow the study of thousands of candidate genes and the identification of those involved in drug efficacy and toxicity. Expression-based genomic technologies such as DNA microarrays and proteomics also facilitate the understanding of important biological and pharmacological pathways, thus identifying more candidate genes for SNP studies. Candidate gene-based pharmacogenetic studies will lead to improved drug development, improved clinical trial design and therapeutics tailored to individual genotypes.

DNA, Complementary↗