Reciprocal regulation of brain and muscle Arnt-like protein 1 and peroxisome proliferator-activated receptor alpha defines a novel positive feedback loop in the rodent liver circadian clock.

Recent evidence has emerged that peroxisome proliferator-activated receptor alpha (PPARalpha), which is largely involved in lipid metabolism, can play an important role in connecting circadian biology and metabolism. In the present study, we investigated the mechanisms by which PPARalpha influences the pacemakers acting in the central clock located in the suprachiasmatic nucleus and in the peripheral oscillator of the liver. We demonstrate that PPARalpha plays a specific role in the peripheral circadian control because it is required to maintain the circadian rhythm of the master clock gene brain and muscle Arnt-like protein 1 (bmal1) in vivo. This regulation occurs via a direct binding of PPARalpha on a potential PPARalpha response element located in the bmal1 promoter. Reversely, BMAL1 is an upstream regulator of PPARalpha gene expression. We further demonstrate that fenofibrate induces circadian rhythm of clock gene expression in cell culture and up-regulates hepatic bmal1 in vivo. Together, these results provide evidence for an additional regulatory feedback loop involving BMAL1 and PPARalpha in peripheral clocks.

Genetic polymorphisms in folate pathway enzymes, DRD4 and GSTM1 are related to temporomandibular disorder

BACKGROUND Temporomandibular disorder (TMD) is a multifactorial syndrome related to a critical period of human life. TMD has been associated with psychological dysfunctions, oxidative state and sexual dimorphism with coincidental occurrence along the pubertal development. In this work we study the association between TMD and genetic polymorphisms of folate metabolism, neurotransmission, oxidative and hormonal metabolism. Folate metabolism, which depends on genes variations and diet, is directly involved in genetic and epigenetic variations that can influence the changes of last growing period of development in human and the appearance of the TMD. METHODS A case-control study was designed to evaluate the impact of genetic polymorphisms above described on TMD. A total of 229 individuals (69% women) were included at the study; 86 were patients with TMD and 143 were healthy control subjects. Subjects underwent to a clinical examination following the guidelines by the Research Diagnostic Criteria for Temporomandibular Disorders (RDC/TMD). Genotyping of 20 Single Nucleotide Polymorphisms (SNPs), divided in two groups, was performed by multiplex minisequencing preceded by multiplex PCR. Other seven genetic polymorphisms different from SNPs (deletions, insertions, tandem repeat, null genotype) were achieved by a multiplex-PCR. A chi-square test was performed to determine the differences in genotype and allelic frequencies between TMD patients and healthy subjects. To estimate TMD risk, in those polymorphisms that shown significant differences, odds ratio (OR) with a 95% of confidence interval were calculated. RESULTS Six of the polymorphisms showed statistical associations with TMD. Four of them are related to enzymes of folates metabolism: Allele G of Serine Hydoxymethyltransferase 1 (SHMT1) rs1979277 (OR = 3.99; 95%CI 1.72, 9.25; p = 0.002), allele G of SHMT1 rs638416 (OR = 2.80; 95%CI 1.51, 5.21; p = 0.013), allele T of Methylentetrahydrofolate Dehydrogenase (MTHFD) rs2236225 (OR = 3.09; 95%CI 1.27, 7.50; p = 0.016) and allele A of Methionine Synthase Reductase (MTRR) rs1801394 (OR = 2.35; 95CI 1.10, 5.00; p = 0.037). An inflammatory oxidative stress enzyme, Gluthatione S-Tranferase Mu-1(GSTM1), null allele (OR = 2.21; 95%CI 1.24, 4.36; p = 0.030) and a neurotransmission receptor, Dopamine Receptor D4 (DRD4), long allele of 48 bp-repeat (OR = 3.62; 95%CI 0.76, 17.26; p = 0.161). CONCLUSIONS Some genetic polymorphisms related to folates metabolism, inflammatory oxidative stress, and neurotransmission responses to pain, has been significantly associated to TMD syndrome.

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.

The biochemistry of drug metabolism-an introduction: part 7. Intra-individual factors affecting drug metabolism.

This review on intra-individual factors affecting drug metabolism completes our series on the biochemistry of drug metabolism. The article presents the molecular mechanisms causing intra-individual differences in enzyme expression and activity. They include enzyme induction by transcriptional activation and enzyme inhibition on the protein level. The influencing factors are of physiological, pathological, or external origin. Tissue characteristics and developmental age strongly influence enzyme-expression patterns. Further influencing factors are pregnancy, disease, or biological rhythms. Xenobiotics, drugs, constituents of herbal remedies, food constituents, ethanol, and tobacco can all influence enzyme expression or activity and, hence, affect drug metabolism.

Steady state plasma levels of the enantiomers of trimipramine and of its metabolites in CYP2D6-, CYP2C19- and CYP3A4/5-phenotyped patients.

Steady state plasma concentrations of the (L)- and (D)-enantiomers of trimipramine (TRI), desmethyltrimipramine (DTRI), 2-hydroxytrimipramine (TRIOH) and 2-hydroxydesmethyl-trimipramine (DTRIOH) were measured in 27 patients receiving between 300 and 400 mg/day racemic TRI. The patients were phenotyped with dextromethorphan and mephenytoin, and the 8-hour urinary ratios of dextromethorphan/dextrorphan, dextromethorphan/3-methoxymorphinan, and (S)-mephenytoin/(R)mephenytoin were used as markers of cytochrome P-450IID6 (CYP2D6), CYP3A4/5 and CYP2C19 activities, respectively. One patient was a CYP2D6 and one was a CYP2C19 poor metabolizer. A stereoselectivity in the metabolism of TRI has been found, with a preferential N-demethylation of (D)-TRI and a preferential hydroxylation of (L)-TRI. CYP2D6 appears to be involved in the 2-hydroxylation of (L)-TRI, (L)DTRI and (D)-DTRI, but not of (D)-TRI, as significant correlations were measured between the dextromethorphan/dextrorphan ratios and the (L)-TRI/(L)-TRIOH (r = 0.45, p = 0.019), the (L)-DTRI/(L)-DTRIOH (r = 0.47, p = 0.014), and the (D)-DTRI/(D)-DTRIOH (r = 0.51, p = 0.006), but not with the (D)-TRI/(D)-TRIOH ratios (r = 0.29, NS). CYP2C19, but not CYP2D6, appears to be involved in the demethylation pathway, with a stereoselectivity toward the (D)-enantiomer of TRI, as a significant positive correlation was calculated between the mephenytoin (S)/(R) ratios and the concentrations to dose-to-weight ratios of (D)-TRI (r = 0.69, p = 0.00006). CYP3A4/5 appears to be involved in the metabolism of (L)-TRI to a presently not determined metabolite. The CYP2D6 poor metabolizer had the highest (L)-DTRI and (D)-DTRI concentrations to dose-to-weight ratios, and the CYP2C19 poor metabolizer had the highest (L)-TRI and (D)-TRI concentrations to dose-to-weight ratios of the group.

In vivo analysis of efavirenz metabolism in individuals with impaired CYP2A6 function.

INTRODUCTION: The antiretroviral drug efavirenz (EFV) is extensively metabolized into three primary metabolites: 8-hydroxy-EFV, 7-hydroxy-EFV and N-glucuronide-EFV. There is a wide interindividual variability in EFV plasma exposure, explained to a great extent by cytochrome P450 2B6 (CYP2B6), the main isoenzyme responsible for EFV metabolism and involved in the major metabolic pathway (8-hydroxylation) and to a lesser extent in 7-hydroxylation. When CYP2B6 function is impaired, the relevance of CYP2A6, the main isoenzyme responsible for 7-hydroxylation may increase. We hypothesize that genetic variability in this gene may contribute to the particularly high, unexplained variability in EFV exposure in individuals with limited CYP2B6 function. METHODS: This study characterized CYP2A6 variation (14 alleles) in individuals (N=169) previously characterized for functional variants in CYP2B6 (18 alleles). Plasma concentrations of EFV and its primary metabolites (8-hydroxy-EFV, 7-hydroxy-EFV and N-glucuronide-EFV) were measured in different genetic backgrounds in vivo. RESULTS: The accessory metabolic pathway CYP2A6 has a critical role in limiting drug accumulation in individuals characterized as CYP2B6 slow metabolizers. CONCLUSION: Dual CYP2B6 and CYP2A6 slow metabolism occurs at significant frequency in various human populations, leading to extremely high EFV exposure.

cAMP-dependent chloride secretion mediates tubule enlargement and cyst formation by cultured mammalian collecting duct cells.

Polycystic kidney diseases result from disruption of the genetically defined program that controls the size and geometry of renal tubules. Cysts which frequently arise from the collecting duct (CD) result from cell proliferation and fluid secretion. From mCCD(cl1) cells, a differentiated mouse CD cell line, we isolated a clonal subpopulation (mCCD-N21) that retains morphogenetic capacity. When grown in three-dimensional gels, mCCD-N21 cells formed highly organized tubular structures consisting of a palisade of polarized epithelial cells surrounding a cylindrical lumen. Subsequent addition of cAMP-elevating agents (forskolin or cholera toxin) or of membrane-permeable cAMP analogs (CPT-cAMP) resulted in rapid and progressive dilatation of existing tubules, leading to the formation of cystlike structures. When grown on filters, mCCD-N21 cells exhibited a high transepithelial resistance as well as aldosterone- and/or vasopressin-induced amiloride-sensitive and -insensitive current. The latter was in part inhibited by Na(+)-K(+)-2Cl(-) cotransporter (bumetanide) and chloride channel (NPPB) inhibitors. Real-time PCR analysis confirmed the expression of NKCC1, the ubiquitous Na(+)-K(+)-2Cl(-) cotransporter and cystic fibrosis transmembrane regulator (CFTR) in mCCD-N21 cells. Tubule enlargement and cyst formation were prevented by inhibitors of Na(+)-K(+)-2Cl(-) cotransporters (bumetanide or ethacrynic acid) or CFTR (NPPB or CFTR inhibitor-172). These results further support the notion that cAMP signaling plays a key role in renal cyst formation, at least in part by promoting chloride-driven fluid secretion. This new in vitro model of tubule-to-cyst conversion affords a unique opportunity for investigating the molecular mechanisms that govern the architecture of epithelial tubes, as well as for dissecting the pathophysiological processes underlying cystic kidney diseases.

Variations of CYP3A activity induced by antiretroviral treatment in HIV-1 infected patients.

OBJECTIVE: To measure the in vivo variations of CYP3A activity induced by anti-HIV drugs in human immunodeficiency virus (HIV)1-positive patients. METHODS: A low oral dose of midazolam (MID) (0.075 mg) was given to the patients and the 30-min total 1-OH midazolam (1-OHMID)/MID ratio was determined. Patients were phenotyped either before the introduction of antiretroviral treatments (control group, 90 patients) or after a variable period of antiretroviral treatment (56 patients). Twenty-one subjects underwent multiple phenotyping tests (before and during the course of the treatment). RESULTS: The median MID ratio was 3.51 in the control group (range 0.20-14.6). It was 5-fold higher in the group with efavirenz (28 patients; median, range: 16.0, 3.81-367; P < 0.0001), 13-fold lower with nelfinavir (18 patients; 0.27, 0.06-36.3; P < 0.0001), 17-fold lower with efavirenz + ritonavir (three patients; 0.21, 0.05-0.47; P = 0.006), 50-fold lower with ritonavir (four patients; 0.07, 0.06-0.17; P = 0.0007), and 7-fold lower with nevirapine + (ritonavir or nelfinavir or grapefruit juice) (three patients; 0.48, 0.03-1.83; P = 0.03). CYP3A activity was lower in the efavirenz + ritonavir group (P = 0.01) and in the ritonavir group (P = 0.04) than in the nelfinavir group, although already strongly inhibited in the latter. CONCLUSION: The low-dose MID phenotyping test was successfully used to measure the in vivo variations of CYP3A activity induced by antiretroviral drugs. Efavirenz strongly induces CYP3A activity, while ritonavir almost completely inhibits it. Nelfinavir strongly decreases CYP3A activity, but to a lesser extent than ritonavir. The inhibition of CYP3A by ritonavir or nelfinavir offsets the inductive effects of efavirenz or nevirapine administered concomitantly. Finally, no induction of CYP3A activity was noticeable after long-term administration of ritonavir at low dosages (200 mg/day b.i.d.) or of nelfinavir at standard dosages (2,500 mg/day b.i.d.).

In vitro biotransformation of imatinib by the tumor expressed CYP1A1 and CYP1B1.

The main objective of the study was to examine the biotransformation of the anticancer drug imatinib in target cells by incubating it with oxidoreductases expressed in tumor cells. The second objective was to obtain an in silico prediction of the potential activity of imatinib metabolites. An in vitro enzyme kinetic study was performed with cDNA expressed human oxidoreductases and LC-MS/MS analysis. The kinetic parameters (Km and Vmax) were determined for six metabolites. A molecular modeling approach was used to dock these metabolites to the target Abl or Bcr-Abl kinases. CYP3A4 isozyme showed the broadest metabolic capacity, whereas CYP1A1, CYP1B1 and FMO3 isozymes biotransformed imatinib with a high intrinsic clearance. The predicted binding modes for the metabolites to Abl were comparable to that of the parent drug, suggesting potential activity. These findings indicate that CYP1A1 and CYP1B1, which are known to be overexpressed in a wide range of tumors, are involved in the biotransformation of imatinib. They could play a role in imatinib disposition in the targeted stem, progenitor and differentiated cancer cells, with a possible contribution of the metabolites toward the activity of the drug.

Oral administration of a low dose of midazolam (75 microg) as an in vivo probe for CYP3A activity.

OBJECTIVE: We investigated whether the oral administration of a low dose (75 micro g) of midazolam, a CYP3A probe, can be used to measure the in vivo CYP3A activity. METHODS: Plasma concentrations of midazolam, 1'OH-midazolam and 4'OH-midazolam were measured after the oral administration of 7.5 mg and 75 micro g midazolam in 13 healthy subjects without medication, in four subjects pretreated for 2 days with ketoconazole (200 mg b.i.d.), a CYP3A inhibitor, and in four subjects pretreated for 4 days with rifampicin (450 mg q.d.), a CYP3A inducer. RESULTS: After oral administration of 75 micro g midazolam, the 30-min total (unconjugated + conjugated) 1'OH-midazolam/midazolam ratios measured in the groups without co-medication, with ketoconazole and with rifampicin were (mean+/-SD): 6.23+/-2.61, 0.79+/-0.39 and 56.1+/-12.4, respectively. No side effects were reported by the subjects taking this low dose of midazolam. Good correlations were observed between the 30-min total 1'OH-midazolam/midazolam ratio and midazolam clearance in the group without co-medication (r(2)=0.64, P<0.001) and in the three groups taken together (r(2)=0.91, P<0.0001). Good correlations were also observed between midazolam plasma levels and midazolam clearance, measured between 1.5 h and 4 h. CONCLUSION: A low oral dose of midazolam can be used to phenotype CYP3A, either by the determination of total 1'OH-midazolam/midazolam ratios at 30 min or by the determination of midazolam plasma levels between 1.5 h and 4 h after its administration.

Routine therapeutic drug monitoring in patients treated with 10-360 mg/day citalopram

From data collected during routine TDM, plasma concentrations of citalopram (CIT) and its metabolites demethylcitalopram (DCIT) and didemethylcitalopram (DDCIT) were measured in 345 plasma samples collected in steady-state conditions. They were from 258 patients treated with usual doses (20-60 mg/d) and from patients medicated with 80-360 mg/d CIT. Most patients had one or several comedications, including other antidepressants, antipsychotics, lithium, anticonvulsants, psychostimulants and somatic medications. Dose-corrected CIT plasma concentrations (C/D ratio) were 2.51 +/- 2.25 ng mL-1 mg-1 (n = 258; mean +/- SD). Patients >65 years had significantly higher dose-corrected CIT plasma concentrations (n = 56; 3.08 +/- 1.35 ng mL-1 mg-1) than younger patients (n = 195; 2.35 +/- 2.46 ng mL-1 mg-1) (P = 0.03). CIT plasma concentrations in the generally recommended dose range were [mean +/- SD, (median)]: 57 +/- 64 (45) ng/mL (10-20 mg/d; n = 64), 117 +/- 95 (91) ng/mL (21-60 mg/d; n = 96). At higher than usual doses, the following concentrations of CIT were measured: 61-120 mg/d CIT, 211 +/- 103 (190) ng/mL (n = 93); 121-200 mg/d: 339 +/- 143 (322) ng/mL (n = 70); 201-280 mg/d: 700 +/- 408 (565) ng/mL (n = 18); 281-360 mg/d: 888 +/- 620 (616) ng/mL (n = 4). When only one sample per patient (at the highest daily dose if repeated dosages) is considered, there is a linear and significant correlation (n = 48, r = 0.730; P < 0.001) between daily dose (10-200 mg/d) and CIT plasma concentrations. In experiments with dogs, DDCIT was reported to affect the QT interval when present at concentrations >300 ng/mL. In this study, DDCIT concentration reached 100 ng/mL in a patient treated with 280 mg/d CIT. Twelve other patients treated with 140-320 mg/d CIT had plasma concentrations of DDCIT within the range 52-73 ng/mL. In a subgroup comprised of patients treated with > or =160 mg/d CIT and with CIT plasma concentrations < or =300 ng/mL, and patients treated with < or =200 mg/d CIT and CIT plasma concentrations > or = 600 ng/mL, the enantiomers of CIT and DCIT were also analyzed. The highest S-CIT concentration measured in this subgroup was 327 ng/mL in a patient treated with 140 mg/d CIT, but the highest S-CIT concentration (632 ng/mL) was measured in patient treated with 360 mg/d CIT. In conclusion, there is a highly linear correlation between CIT plasma concentrations and CIT doses, well above the usual dose range.

Genetic removal of tri-unsaturated fatty acids suppresses developmental and molecular phenotypes of an Arabidopsis tocopherol-deficient mutant. Whole-body mapping of malondialdehyde pools in a complex eukaryote.

Malondialdehyde (MDA) is a small, ubiquitous, and potentially toxic aldehyde that is produced in vivo by lipid oxidation and that is able to affect gene expression. Tocopherol deficiency in the vitamin E2 mutant vte2-1 of Arabidopsis thaliana leads to massive lipid oxidation and MDA accumulation shortly after germination. MDA accumulation correlates with a strong visual phenotype (growth reduction, cotyledon bleaching) and aberrant GST1 (glutathione S-transferase 1) expression. We suppressed MDA accumulation in the vte2-1 background by genetically removing tri-unsaturated fatty acids. The resulting quadruple mutant, fad3-2 fad7-2 fad8 vte2-1, did not display the visual phenotype or the aberrant GST1 expression observed in vte2-1. Moreover, cotyledon bleaching in vte2-1 was chemically phenocopied by treatment of wild-type plants with MDA. These data suggest that products of tri-unsaturated fatty acid oxidation underlie the vte2-1 seedling phenotype, including cellular toxicity and gene regulation properties. Generation of the quadruple mutant facilitated the development of an in situ fluorescence assay based on the formation of adducts of MDA with 2-thiobarbituric acid at 37 degrees C. Specificity was verified by measuring pentafluorophenylhydrazine derivatives of MDA and by liquid chromatography analysis of MDA-2-thiobarbituric acid adducts. Potentially applicable to other organisms, this method allowed the localization of MDA pools throughout the body of Arabidopsis and revealed an undiscovered pool of the compound unlikely to be derived from trienoic fatty acids in the vicinity of the root tip quiescent center.

Protection of Structural Concrete Substructures, HR-220, December 1992

In the past, many pier columns were deteriorating due to attack by chlorides. The chloride (from deicers) has attacked the substructures by drainage from the superstructure. Piers supporting grade separation bridges are also subject to chlorides contained in the direct splash from lower level traffic. Repairs of these piers are both difficult and costly. In this project, four different sealants were applied to piers to evaluate their use in the protection of the concrete against chloride-ions. One pier was left untreated to use as a control pier with which to compare the protected piers. This project began in 1980 and was to be completed in 1985, but at that time it was determined further testing was needed to make a more conclusive evaluation.

Mouse GLUT9: evidences for a urate uniporter.

GLUT9 (SLC2A9) is a newly described urate transporter whose function, characteristics, and localization have just started to be elucidated. Some transport properties of human GLUT9 have been studied in the Xenopus laevis oocyte expression system, but the type of transport (uniport, coupled transport system, stoichiometry ... .) is still largely unknown. We used the same experimental system to characterize in more detail the transport properties of mouse GLUT9, its sensitivity to several uricosuric drugs, and the specificities of two splice variants, mGLUT9a and mGLUT9b. [(14)C]urate uptake measurements show that both splice variants are high-capacity urate transporters and have a K(m) of approximately 650 microM. The well-known uricosuric agents benzbromarone (500 microM) and losartan (1 mM) inhibit GLUT9-mediated urate uptake by 90 and 50%, respectively. Surprisingly, phloretin, a glucose-transporter blocker, inhibits [(14)C]urate uptake by approximately 50% at 1 mM. Electrophysiological measurements suggest that urate transport by mouse GLUT9 is electrogenic and voltage dependent, but independent of the Na(+) and Cl(-) transmembrane gradients. Taken together, our results suggest that GLUT9 works as a urate (anion) uniporter. Finally, we show by RT-PCR performed on RNA from mouse kidney microdissected tubules that GLUT9a is expressed at low levels in proximal tubules, while GLUT9b is specifically expressed in distal convoluted and connecting tubules. Expression of mouse GLUT9 in the kidney differs from that of human GLUT9, which could account for species differences in urate handling.

The circadian PAR-domain basic leucine zipper transcription factors DBP, TEF, and HLF modulate basal and inducible xenobiotic detoxification.

The PAR-domain basic leucine zipper (PAR bZip) transcription factors DBP, TEF, and HLF accumulate in a highly circadian manner in several peripheral tissues, including liver and kidney. Mice devoid of all three of these proteins are born at expected Mendelian ratios, but are epilepsy prone, age at an accelerated rate, and die prematurely. In the hope of identifying PAR bZip target genes whose altered expression might contribute to the high morbidity and mortality of PAR bZip triple knockout mice, we compared the liver and kidney transcriptomes of these animals to those of wild-type or heterozygous mutant mice. These experiments revealed that PAR bZip proteins control the expression of many enzymes and regulators involved in detoxification and drug metabolism, such as cytochrome P450 enzymes, carboxylesterases, and constitutive androstane receptor (CAR). Indeed, PAR bZip triple knockout mice are hypersensitive to xenobiotic compounds, and the deficiency in detoxification may contribute to their early aging.