Cholesterol 24S-Hydroxylase overexpression inhibits the liver X receptor (LXR) pathway by activating small guanosine triphosphate-binding proteins (sGTPases) in neuronal cells

The neuronal-specific cholesterol 24S-hydroxylase (CYP46A1) is important for brain cholesterol elimination. Cyp46a1 null mice exhibit severe deficiencies in learning and hippocampal long-term potentiation, suggested to be caused by a decrease in isoprenoid intermediates of the mevalonate pathway. Conversely, transgenic mice overexpressing CYP46A1 show an improved cognitive function. These results raised the question of whether CYP46A1 expression can modulate the activity of proteins that are crucial for neuronal function, namely of isoprenylated small guanosine triphosphate-binding proteins (sGTPases). Our results show that CYP46A1 overexpression in SH-SY5Y neuroblastoma cells and in primary cultures of rat cortical neurons leads to an increase in 3-hydroxy-3-methyl-glutaryl-CoA reductase activity and to an overall increase in membrane levels of RhoA, Rac1, Cdc42 and Rab8. This increase is accompanied by a specific increase in RhoA activation. Interestingly, treatment with lovastatin or a geranylgeranyltransferase-I inhibitor abolished the CYP46A1 effect. The CYP46A1-mediated increase in sGTPases membrane abundance was confirmed in vivo, in membrane fractions obtained from transgenic mice overexpressing this enzyme. Moreover, CYP46A1 overexpression leads to a decrease in the liver X receptor (LXR) transcriptional activity and in the mRNA levels of ATP-binding cassette transporter 1, sub-family A, member 1 and apolipoprotein E. This effect was abolished by inhibition of prenylation or by co-transfection of a RhoA dominant-negative mutant. Our results suggest a novel regulatory axis in neurons; under conditions of membrane cholesterol reduction by increased CYP46A1 expression, neurons increase isoprenoid synthesis and sGTPase prenylation. This leads to a reduction in LXR activity, and consequently to a decrease in the expression of LXR target genes.

A farmacogenética e a medicina personalizada

A farmacogenética tem por objetivo a identificação de diferenças genéticas entre indivíduos que possam influenciar a resposta à terapêutica farmacológica, melhorando a sua eficácia e segurança. Associado à farmacogenética surge a “medicina personalizada”, ou seja, em oposição à existência de um fármaco que consiga tratar todos os pacientes, o tratamento individualizado parece o caminho mais promissor, uma vez que reduz o risco de reações adversas por toxicidade (segurança), adequa a dose ao indivíduo, evitando excessos ou défices (dose) e evita a metodologia de tentativa erro na escolha do fármaco (eficácia). A farmacogenética é relevante para a resposta individual ao fármaco por duas vias distintas: a farmacocinética e a farmacodinâmica. A variabilidade genética pode afetar a forma como um fármaco pode ser absorvido, ativado, metabolizado ou excretado, podendo conduzir assim a uma variabilidade na resposta. De entre o número infindável de possíveis exemplos, nesta revisão apresentam-se exemplos relacionados com os genes do Citocromo P450, do gene NAT2 e do gene da Colinesterase. As diferenças genéticas entre os indivíduos podem ainda afetar a resposta ao fármaco pela sua farmacodinâmica, ou seja, a resposta específica do alvo ao fármaco. De entre a multiplicidade de alvos de fármacos existentes serão apresentados exemplos do gene da G6PD e do VKORC1. Apesar de alguns dados científicos indicarem benefício para o paciente, ainda está longe de a farmacogenética fazer parte da prática clínica de rotina, talvez porque os custos-benefícios ainda não foram avaliados de forma precisa.

DNA nanoprobes for molecular detection

Dissertação apresentada para obtenção do Grau de Doutor em Engenharia Biológica – especialidade Engenharia Genética, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Response to antiretroviral treatment in HIV-1-infected individuals with allelic variants of the multidrug resistance transporter 1: a pharmacogenetics study.

BACKGROUND:HIV-1-infected patients vary considerably by their response to antiretroviral treatment, drug concentrations in plasma, toxic events, and rate of immune recovery. This variability could have a genetic basis. We did a pharmacogenetics study to analyse the association between response to antiretroviral treatment and allelic variants of several genes. METHODS:In 123 patients, we did PCR analyses of the gene for the multidrug-resistance transporter (MDR1), which codes for P-glycoprotein, of genes coding for isoenzymes of cytochrome P450, CYP3A4, CYP3A5, CYP2D6, and CYP2C19, and of the gene for the chemokine receptor CCR5. We measured concentrations in plasma of the antiretroviral agents efavirenz and nelfinavir by high-performance liquid-chromatography, and measured levels of P-glycoprotein expression, CD4-cell count, and HIV-1 viraemia. FINDINGS: Median drug concentrations in patients with the MDR1 3435 TT, CT, and CC genotypes were at the 30th, 50th, and 75th percentiles, respectively (p=0.0001). In patients with CYP2D6 extensive-metaboliser or poor-metaboliser alleles, median drug concentrations were at percentiles 45 and 62.5, respectively (p=0.04). Patients with the MDR1 TT genotype 6 months after starting treatment had a greater rise in CD4-cell count (257 cells/microL) than patients with the CT (165 cells/microL) and CC (121 cells/microL) genotype (p=0.0048), and the best recovery of naïve CD4-cells. INTERPRETATION:The polymorphism MDR1 3435 C/T predicts immune recovery after initiation of antiretroviral treatment. This finding suggests that P-glycoprotein has an important role in admittance of antiretroviral drugs to restricted compartments in vivo.

Effects of grapefruit juice on the pharmacokinetics of the enantiomers of methadone

BACKGROUND AND OBJECTIVES: Cytochrome P450 (CYP) 3A4 is the main CYP isozyme involved in methadone metabolism. We investigated the influence of grapefruit juice, which contains inhibitors of intestinal CYP3A, on the steady-state pharmacokinetics of methadone. METHODS: For 5 days, 8 patients undergoing methadone maintenance treatment received 200 mL water or grapefruit juice 30 minutes before and again together with their daily dose of methadone. Blood sampling for R-, S-, and R,S-methadone plasma determination was performed over a 24-hour period. CYP3A activity was determined by measuring the plasma 1'-hydroxymidazolam/midazolam ratio. RESULTS: A decrease in the midazolam ratio was measured in all patients after grapefruit juice (mean +/- SD before grapefruit juice, 9.3 +/- 5.9; mean +/- SD after grapefruit juice, 3.9 +/- 1.2; P <.05). Grapefruit juice led to a mean 17% increase in the area under the curve extrapolated to 24 hours for both enantiomers of methadone (range, 3% to 29% [P <.005]; range, -4% to 37% [P <.05]; and range, 1% to 32% [P <.01]; for R-, S-, and R,S-methadone, respectively). A similar increase in peak level and decrease in apparent clearance were measured with grapefruit juice, whereas time to peak level, terminal half-life, and apparent volume during the terminal phase of R-, S-, and R,S-methadone were not affected by grapefruit juice. No symptom of overmedication was either detected by the clinical staff or reported by the patients. CONCLUSIONS: Grapefruit juice administration is associated with a modest increase in methadone bioavailability, which is not expected to endanger patients. However, it cannot be excluded that a much stronger effect may occur in some patients, and thus grapefruit juice intake is not recommended during methadone maintenance treatment, in particular in patients initiating such a treatment.

Phase I and phase II xenobiotic reactions and metabolism of the food-borne carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in aggregating liver cell cultures.

Aggregating fetal liver cell cultures were tested for their ability to metabolize xenobiotics using ethoxycoumarin-O-deethylase (ECOD), as marker of phase I metabolism, and glutathione S-transferase (GST), as marker for phase II reactions. Significant basal activities, stable over 14 days in culture were measured for both ECOD and GST activities. The prototype cytochrome P450 inducers, 3-methylcholanthrene (3-MC) and phenobarbital (PB), increased ECOD and GST activities reaching an optimum 7 days after culturing, followed by a decline in activity. This decline was partially prevented by 1% dimethyl sulfoxide (DMSO) added chronically to the culture medium. DMSO was also found to induce ECOD activity and to a lesser extent GST activity. Furthermore, it potentiated in a dose-dependent manner the induction of ECOD by PB. The food-borne carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) is metabolically transformed through a number of pathways in vivo. It was therefore used to examine the metabolic capacity in fetal and adult liver cell aggregates. Metabolism of MeIQx was mainly through N2-conjugation, resulting in formation of the N2-glucuronide and sulfamate conjugates for non-induced fetal liver cells. These metabolites were also found in large amounts in non-induced adult liver cells. Low levels of cytochrome P450-mediated ring-hydroxylated metabolites were detected in both non-induced fetal and adult liver cells. After induction with arochlor (PCB) or 3-MC, the major pathway was ring-hydroxylation (cytochrome P450 dependent), followed by conjugation to beta-glucuronic or sulfuric acid. The presence of the glucuronide conjugate of N-hydroxy-MeIQx, a mutagenic metabolite, suggested an induction of P450 CYP1A2. The metabolism of MeIQx by liver cell aggregates is very similar to that observed in vivo and suggests that aggregating liver cell cultures are a useful model for in vitro metabolic studies in toxicology.

Association of CYP3A5 genotypes with blood pressure and renal function in African families.

OBJECTIVE: Renal cytochrome P450 3A5 (CYP3A5) activity has been associated with blood pressure and salt sensitivity in humans. We determined whether CYP3A5 polymorphisms are associated with ambulatory blood pressure (ABP) and with glomerular filtration rate (GFR) in African families. METHODS: Using a cross-sectional design, 375 individuals from 72 families, each with at least two hypertensive siblings, were recruited through a hypertension register in the Seychelles (Indian Ocean). We analyzed the association between the CYP3A5 alleles (*1, *3, *6 and *7) and ABP, GFR and renal sodium handling (fractional excretion of lithium), from pedigree data, allowing for other covariates and familial correlations. RESULTS: CYP3A5*1 carriers increased their daytime systolic and diastolic ABP with age (0.55 and 0.23 mmHg/year) more than non-carriers (0.21 and 0.04 mmHg/year). CYP3A5*1 had a significant main effect on daytime systolic/diastolic ABP [regression coefficient (SE): -29.6 (10.0)/-8.2 (4.1) mmHg, P = 0.003/0.045, respectively] and this effect was modified by age (CYP3A5*1 x age interactions, P = 0.017/0.018). For night-time ABP, the effect of CYP3A5*1 was modified by urinary sodium excretion, not by age. For renal function, CYP3A5*1 carriers had a 7.6(3.8) ml/min lower GFR (P = 0.045) than non-carriers. Proximal sodium reabsorption decreased with age in non-carriers, but not in CYP3A5*1 carriers (P for interaction = 0.02). CONCLUSIONS: These data demonstrate that CYP3A5 polymorphisms are associated with ambulatory BP, CYP3A5*1 carriers showing a higher age- and sodium- related increase in ABP than non-carriers. The age effect may be due, in part, to the action of CYP3A5 on renal sodium handling.

Role of enhanced detoxication in a deltamethrin-resistant population of Triatoma infestans (Hemiptera, Reduviidae) from Argentina

Deltamethrin and other pyrethroids have been extensively used in Argentina since 1980, for the chemical control of Triatoma infestans Klug (Hemiptera: Reduviidae). Recently, resistance to deltamethrin was detected in field populations by the survival of bugs exposed by topical application to the diagnostic dose estimated on the CIPEIN susceptible strain. Results of the current study showed low resistant ratios (RRs) to deltamethrin for the resistant populations (RR ranged from 2.0 for San Luis colony to 7.9 for Salta colony). Biochemical studies were made on the most resistant colony (Salta) and the susceptible strain (CIPEIN), in order to establish the importance of degradative mechanisms as a cause of the detected resistance. Esterase activity was measured on 3 days old first instars through phenylthioacetate and a-naphtyl acetate activities. The results showed a significant difference in no cholinesterase esterase activity from susceptible (7.6 ± 0,7 µM S./i.min.) and Salta resistant colony (9.5 ± 0.8 µM S./i.min.). Cytochrome P450 mono-oxygenase (P450) activity was measured on individual insects through ethoxycoumarine deethylase (ECOD) activity using a fluorescence micro plate reader. The dependence of ECOD activity on age and body region of the nymphs, and pH and time of incubation were studied in order to optimize the measurement. As a result, comparative studies were performed on abdomens of 2 days old first instars at pH 7.2 and 4 h incubation time. ECOD activity of first nymphs was significantly lower in the susceptible colony (61.3 ± 9.08 pg ECOD/ insect) than in the resistant one (108.1± 5.7 pg ECOD/ insect). These results suggest that degradative esterases (no-cholinesterase) and mono-oxygenases cytochrome P450, play an important role in the resistance to deltamethrin in Salta colony from Argentina.

Draft genome of the globally widespread and invasive Argentine ant (Linepithema humile).

Ants are some of the most abundant and familiar animals on Earth, and they play vital roles in most terrestrial ecosystems. Although all ants are eusocial, and display a variety of complex and fascinating behaviors, few genomic resources exist for them. Here, we report the draft genome sequence of a particularly widespread and well-studied species, the invasive Argentine ant (Linepithema humile), which was accomplished using a combination of 454 (Roche) and Illumina sequencing and community-based funding rather than federal grant support. Manual annotation of >1,000 genes from a variety of different gene families and functional classes reveals unique features of the Argentine ant's biology, as well as similarities to Apis mellifera and Nasonia vitripennis. Distinctive features of the Argentine ant genome include remarkable expansions of gustatory (116 genes) and odorant receptors (367 genes), an abundance of cytochrome P450 genes (>110), lineage-specific expansions of yellow/major royal jelly proteins and desaturases, and complete CpG DNA methylation and RNAi toolkits. The Argentine ant genome contains fewer immune genes than Drosophila and Tribolium, which may reflect the prominent role played by behavioral and chemical suppression of pathogens. Analysis of the ratio of observed to expected CpG nucleotides for genes in the reproductive development and apoptosis pathways suggests higher levels of methylation than in the genome overall. The resources provided by this genome sequence will offer an abundance of tools for researchers seeking to illuminate the fascinating biology of this emerging model organism.

Two approaches to discovering and developing new drugs for Chagas disease

This review will focus on two general approaches carried out at the Sandler Center, University of California, San Francisco, to address the challenge of developing new drugs for the treatment of Chagas disease. The first approach is target-based drug discovery, and two specific targets, cytochrome P450 CYP51 and cruzain (aka cruzipain), are discussed. A "proof of concept" molecule, the vinyl sulfone inhibitor K777, is now a clinical candidate. The preclinical assessment compliance for filing as an Investigational New Drug with the United States Food and Drug Administration (FDA) is presented, and an outline of potential clinical trials is given. The second approach to identifying new drug leads is parasite phenotypic screens in culture. The development of an assay allowing high throughput screening of Trypanosoma cruzi amastigotes in skeletal muscle cells is presented. This screen has the advantage of not requiring specific strains of parasites, so it could be used with field isolates, drug resistant strains or laboratory strains. It is optimized for robotic liquid handling and has been validated through a screen of a library of FDA-approved drugs identifying 65 hits.

Characterization of microevolution events in Mycobacterium tuberculosis strains involved in recent transmission clusters.

Under certain circumstances, it is possible to identify clonal variants of Mycobacterium tuberculosis infecting a single patient, probably as a result of subtle genetic rearrangements in part of the bacillary population. We systematically searched for these microevolution events in a different context, namely, recent transmission chains. We studied the clustered cases identified using a population-based universal molecular epidemiology strategy over a 5-year period. Clonal variants of the reference strain defining the cluster were found in 9 (12%) of the 74 clusters identified after the genotyping of 612 M. tuberculosis isolates by IS6110 restriction fragment length polymorphism analysis and mycobacterial interspersed repetitive units-variable-number tandem repeat typing. Clusters with microevolution events were epidemiologically supported and involved 4 to 9 cases diagnosed over a 1- to 5-year period. The IS6110 insertion sites from 16 representative isolates of reference and microevolved variants were mapped by ligation-mediated PCR in order to characterize the genetic background involved in microevolution. Both intragenic and intergenic IS6110 locations resulted from these microevolution events. Among those cases of IS6110 locations in intergenic regions which could have an effect on the regulation of adjacent genes, we identified the overexpression of cytochrome P450 in one microevolved variant using quantitative real-time reverse transcription-PCR. Our results help to define the frequency with which microevolution can be expected in M. tuberculosis transmission chains. They provide a snapshot of the genetic background of these subtle rearrangements and identify an event in which IS6110-mediated microevolution in an isogenic background has functional consequences.

Insights into the transcriptome of oenocytes from Aedes aegypti pupae

Oenocytes are ectodermic cells present in the fat body of several insect species and these cells are considered to be analogous to the mammalian liver, based on their role in lipid storage, metabolism and secretion. Although oenocytes were identified over a century ago, little is known about their messenger RNA expression profiles. In this study, we investigated the transcriptome of Aedes aegypti oenocytes. We constructed a cDNA library from Ae. aegypti MOYO-R strain oenocytes collected from pupae and randomly sequenced 687 clones. After sequences editing and assembly, 326 high-quality contigs were generated. The most abundant transcripts identified corresponded to the cytochrome P450 superfamily, whose members have roles primarily related to detoxification and lipid metabolism. In addition, we identified 18 other transcripts with putative functions associated with lipid metabolism. One such transcript, a fatty acid synthase, is highly represented in the cDNA library of oenocytes. Moreover, oenocytes expressed several immunity-related genes and the majority of these genes were lysozymes. The transcriptional profile suggests that oenocytes play diverse roles, such as detoxification and lipid metabolism, and increase our understanding of the importance of oenocytes in Ae. aegypti homeostasis and immune competence.

Linee guida della consensus conference dell'ANGP per il monitoraggio plasmatico dei farmaci (TDM) in psichiatria. Aggiornamento 2011 =AGNP consensus guidelines for therapeutic drug monitoring in psychiatry: update 2011

Therapeutic drug monitoring (TDM), i.e., the quantification of serum or plasma concentrations of medications for dose optimization, has proven a valuable tool for the patient-matched psychopharmacotherapy. Uncertain drug adherence, suboptimal tolerability, non-response at therapeutic doses, or pharmacokinetic drug-drug interactions are typical situations when measurement of medication concentrations is helpful. Patient populations that may predominantly benefit from TDM in psychiatry are children, pregnant women, elderly patients, individuals with intelligence disabilities, forensic patients, patients with known or suspected genetically determined pharmacokinetic abnormalities or individuals with pharmacokinetically relevant comorbidities. However, the potential benefits of TDM for optimization of pharmacotherapy can only be obtained if the method is adequately integrated into the clinical treatment process. To promote an appropriate use of TDM, the TDM expert group of the Arbeitsgemeinschaft für Neuropsychopharmakologie und Pharmakopsychiatrie (AGNP) issued guidelines for TDM in psychiatry in 2004. Since then, knowledge has advanced significantly, and new psychopharmacologic agents have been introduced that are also candidates for TDM. Therefore the TDM consensus guidelines were updated and extended to 128 neuropsychiatric drugs. 4 levels of recommendation for using TDM were defined ranging from "strongly recommended" to "potentially useful". Evidence-based "therapeutic reference ranges" and "dose related reference ranges" were elaborated after an extensive literature search and a structured internal review process. A "laboratory alert level" was introduced, i.e., a plasma level at or above which the laboratory should immediately inform the treating physician. Supportive information such as cytochrome P450 substrateand inhibitor properties of medications, normal ranges of ratios of concentrations of drug metabolite to parent drug and recommendations for the interpretative services are given. Recommendations when to combine TDM with pharmacogenetic tests are also provided. Following the guidelines will help to improve the outcomes of psychopharmacotherapy of many patients especially in case of pharmacokinetic problems. Thereby, one should never forget that TDM is an interdisciplinary task that sometimes requires the respectful discussion of apparently discrepant data so that, ultimately, the patient can profit from such a joint effort.

The effects of CYP2D6 and CYP3A activities on the pharmacokinetics of immediate release oxycodone.

BACKGROUND AND PURPOSE: There is high interindividual variability in the activity of drug-metabolizing enzymes catalysing the oxidation of oxycodone [cytochrome P450 (CYP) 2D6 and 3A], due to genetic polymorphisms and/or drug-drug interactions. The effects of CYP2D6 and/or CYP3A activity modulation on the pharmacokinetics of oxycodone remains poorly explored. EXPERIMENTAL APPROACH: A randomized crossover double-blind placebo-controlled study was performed with 10 healthy volunteers genotyped for CYP2D6 [six extensive (EM), two deficient (PM/IM) and two ultrarapid metabolizers (UM)]. The volunteers randomly received on five different occasions: oxycodone 0.2 mg x kg(-1) and placebo; oxycodone and quinidine (CYP2D6 inhibitor); oxycodone and ketoconazole (CYP3A inhibitor); oxycodone and quinidine+ketoconazole; placebo. Blood samples for plasma concentrations of oxycodone and metabolites (oxymorphone, noroxycodone and noroxymorphone) were collected for 24 h after dosing. Phenotyping for CYP2D6 (with dextromethorphan) and CYP3A (with midazolam) were assessed at each session. KEY RESULTS: CYP2D6 activity was correlated with oxymorphone and noroxymorphone AUCs and C(max) (-0.71 < Spearman correlation coefficient rhos < -0.92). Oxymorphone C(max) was 62% and 75% lower in PM than EM and UM. Noroxymorphone C(max) reduction was even more pronounced (90%). In UM, oxymorphone and noroxymorphone concentrations increased whereas noroxycodone exposure was halved. Blocking CYP2D6 (with quinidine) reduced oxymorphone and noroxymorphone C(max) by 40% and 80%, and increased noroxycodone AUC(infinity) by 70%. Blocking CYP3A4 (with ketoconazole) tripled oxymorphone AUC(infinity) and reduced noroxycodone and noroxymorphone AUCs by 80%. Shunting to CYP2D6 pathway was observed after CYP3A4 inhibition. CONCLUSIONS AND IMPLICATIONS: Drug-drug interactions via CYP2D6 and CYP3A affected oxycodone pharmacokinetics and its magnitude depended on CYP2D6 genotype.

An autoregulatory loop controlling CYP1A1 gene expression: role of H(2)O(2) and NFI.

Cytochrome P450 1A1 (CYP1A1), like many monooxygenases, can produce reactive oxygen species during its catalytic cycle. Apart from the well-characterized xenobiotic-elicited induction, the regulatory mechanisms involved in the control of the steady-state activity of CYP1A1 have not been elucidated. We show here that reactive oxygen species generated from the activity of CYP1A1 limit the levels of induced CYP1A1 mRNAs. The mechanism involves the repression of the CYP1A1 gene promoter activity in a negative-feedback autoregulatory loop. Indeed, increasing the CYP1A1 activity by transfecting CYP1A1 expression vectors into hepatoma cells elicited an oxidative stress and led to the repression of a reporter gene driven by the CYP1A1 gene promoter. This negative autoregulation is abolished by ellipticine (an inhibitor of CYP1A1) and by catalase (which catalyzes H(2)O(2) catabolism), thus implying that H(2)O(2) is an intermediate. Down-regulation is also abolished by the mutation of the proximal nuclear factor I (NFI) site in the promoter. The transactivating domain of NFI/CTF was found to act in synergy with the arylhydrocarbon receptor pathway during the induction of CYP1A1 by 2,3,7,8-tetrachloro-p-dibenzodioxin. Using an NFI/CTF-Gal4 fusion, we show that NFI/CTF transactivating function is decreased by a high activity of CYP1A1. This regulation is also abolished by catalase or ellipticine. Consistently, the transactivating function of NFI/CTF is repressed in cells treated with H(2)O(2), a novel finding indicating that the transactivating domain of a transcription factor can be targeted by oxidative stress. In conclusion, an autoregulatory loop leads to the fine tuning of the CYP1A1 gene expression through the down-regulation of NFI activity by CYP1A1-based H(2)O(2) production. This mechanism allows a limitation of the potentially toxic CYP1A1 activity within the cell.