Pharmacometabolomic mapping of early biochemical changes induced by sertraline and placebo.

In this study, we characterized early biochemical changes associated with sertraline and placebo administration and changes associated with a reduction in depressive symptoms in patients with major depressive disorder (MDD). MDD patients received sertraline or placebo in a double-blind 4-week trial; baseline, 1 week, and 4 weeks serum samples were profiled using a gas chromatography time of flight mass spectrometry metabolomics platform. Intermediates of TCA and urea cycles, fatty acids and intermediates of lipid biosynthesis, amino acids, sugars and gut-derived metabolites were changed after 1 and 4 weeks of treatment. Some of the changes were common to the sertraline- and placebo-treated groups. Changes after 4 weeks of treatment in both groups were more extensive. Pathway analysis in the sertraline group suggested an effect of drug on ABC and solute transporters, fatty acid receptors and transporters, G signaling molecules and regulation of lipid metabolism. Correlation between biochemical changes and treatment outcomes in the sertraline group suggested a strong association with changes in levels of branched chain amino acids (BCAAs), lower BCAAs levels correlated with better treatment outcomes; pathway analysis in this group revealed that methionine and tyrosine correlated with BCAAs. Lower levels of lactic acid, higher levels of TCA/urea cycle intermediates, and 3-hydroxybutanoic acid correlated with better treatment outcomes in placebo group. Results of this study indicate that biochemical changes induced by drug continue to evolve over 4 weeks of treatment and that might explain partially delayed response. Response to drug and response to placebo share common pathways but some pathways are more affected by drug treatment. BCAAs seem to be implicated in mechanisms of recovery from a depressed state following sertraline treatment.

Pretreatment metabotype as a predictor of response to sertraline or placebo in depressed outpatients: a proof of concept.

The purpose of this study was to determine whether the baseline metabolic profile (that is, metabotype) of a patient with major depressive disorder (MDD) would define how an individual will respond to treatment. Outpatients with MDD were randomly assigned to sertraline (up to 150 mg per day) (N=43) or placebo (N=46) in a double-blind 4-week trial. Baseline serum samples were profiled using the liquid chromatography electrochemical array; the output was digitized to create a 'digital map' of the entire measurable response for a particular sample. Response was defined as ≥50% reduction baseline to week 4 in the 17-item Hamilton Rating Scale for Depression total score. Models were built using the one-out method for cross-validation. Multivariate analyses showed that metabolic profiles partially separated responders and non-responders to sertraline or to placebo. For the sertraline models, the overall correct classification rate was 81% whereas it was 72% for the placebo models. Several pathways were implicated in separation of responders and non-responders on sertraline and on placebo including phenylalanine, tryptophan, purine and tocopherol. Dihydroxyphenylacetic acid, tocopherols and serotonin were common metabolites in separating responders and non-responders to both drug and placebo. Pretreatment metabotypes may predict which depressed patients will respond to acute treatment (4 weeks) with sertraline or placebo. Some pathways were informative for both treatments whereas other pathways were unique in predicting response to either sertraline or placebo. Metabolomics may inform the biochemical basis for the early efficacy of sertraline.

Application of magnetic resonance microscopy to tissue engineering: a polylactide model.

Absorbable polymers are unique materials that find application as temporary scaffolds in tissue engineering. They are often extremely sensitive to histological processing and, for this reason, studying fragile, tissue-engineered constructs before implantation can be quite difficult. This research investigates the use of noninvasive imaging using magnetic resonance microscopy (MRM) as a tool to enhance the assessment of these cellular constructs. A series of cellular, polylactide constructs was developed and analyzed using a battery of tests, including MRM. Distribution of rat aortic smooth muscle cells within the scaffolds was compared as one example of a tissue engineering MRM application. Cells were loaded in varying amounts using static and dynamic methods. It was found that the cellular component was readily identified and the polymer microstructure readily assessed. Specifically, the MRM results showed a heterogeneous distribution of cells due to static loading and a homogenous distribution associated with dynamic loading, results that were not visible through biochemical tests, scanning electron microscopy, or histological evaluation independently. MRM also allowed differentiation between different levels of cellular loading. The current state of MRM is such that it is extremely useful in the refinement of polymer processing and cell seeding methods. This method has the potential, with technological advances, to be of future use in the characterization of cell-polymer interactions.

Virtual neuropathology: three-dimensional visualization of lesions due to toxic insult.

A first-pass approach incorporating high-field magnetic resonance imaging (MRI) was used for rapid detection of neuropathologic lesions in fixed rat brains. This inherently 3-dimensional and nondestructive technique provides high-resolution, high-contrast images of fixed neuronal tissue in the absence of sectioning or staining. This technique, magnetic resonance microscopy (MRM), was used to identify diverse lesions in 2 well-established rat neurotoxicity models. The intrinsic contrast in the images delineated lesions that were identified using a battery of histologic stains, some of which would not be used in routine screening. Furthermore, the MRM images provided the locations of lesions, which were verified upon subsequent sectioning and staining of the same samples. The inherent contrast generated by water properties is exploited in MRM by choosing suitable pulse sequences, or proton stains. This approach provides the potential for a comprehensive initial MRM screen for neurotoxicity in preclinical models with the capability for extrapolation to clinical analyses using classical MRI.

Abnormal water metabolism in mice lacking the type 1A receptor for ANG II.

Mice lacking AT(1A) receptors for ANG II have a defect in urinary concentration manifested by an inability to increase urinary osmolality to levels seen in controls after thirsting. This defect results in extreme serum hypertonicity during water deprivation. In the basal state, plasma vasopressin levels are similar in wild-type controls and Agtr1a -/- mice. Plasma vasopressin levels increase normally in the AT(1A) receptor-deficient mice after 24 h of water deprivation, suggesting that the defect in urine concentration is intrinsic to the kidney. Using magnetic resonance microscopy, we find that the absence of AT(1A) receptors is associated with a modest reduction in the distance from the kidney surface to the tip of the papilla. However, this structural abnormality seems to play little role in the urinary concentrating defect in Agtr1a -/- mice since the impairment is largely reproduced in wild-type mice by treatment with an AT(1)-receptor antagonist. These studies demonstrate a critical role for the AT(1A) receptor in maintaining inner medullary structures in the kidney and in regulating renal water excretion.

Magnetic resonance microscopy and histopathology: comparative approach of bromobenzene-induced hepatotoxicity in the rat.

The development of magnetic resonance (MR) microscopy has provided new approaches to histology and histopathology. Recent work has shown the promise of increased sensitivity in animal models of chemically induced hepatotoxicity. However, the field is so new that there is little experience to relate changes seen in MR micrographs to the more traditional optical images stained with hematoxylin and eosin. This work compares the sensitivity and reproducibility of MR microscopy with conventional histopathology in detecting bromobenzene-induced hepatotoxicity in the rat. A time-course study was undertaken to provide a range of histopathologies. Specimens were studied at 24, 48, 72, and 96 hours after exposure to 10% of the median lethal dose of bromobenzene. Using 4 animals per group (a total of 32 rats) added statistical significance to the study and defined a range of interanimal variability over 96 hours. This work shows that MR microscopy, besides being nondestructive and three-dimensional, is at least as sensitive as conventional hematoxylin-eosin staining in detecting bromobenzene-induced centrilobular lesions and recovery of the hepatocellular architecture in the rat. This study further suggests that, as we begin to understand the underlying mechanisms of contrast in MR histology, MR may, in fact, supply even higher specificity than more traditional studies: variations were observed in MR images of treated livers at a given time point that could be not be differentiated based on the grading of necrosis and inflammation on hematoxylin-eosin-stained sections.

Mono- and dimethylation of arsenic in rat liver cytosol in vitro.

Production of methylarsonate and dimethylarsinate from radiolabelled [73 As]arsenite and [73 As]arsenate was examined in an assay system that contained cytosol prepared from a 20% homogenate (w/v) of livers from 8- 10-week-old male Fischer 344 rats. After a 60-min incubation at 37 degrees C with added S-adenosylmethionine and glutathione, up to 50% of carrier-free [73As]arsenite and about 15% of carrier-free [73As]arsenate were methylated. Incubation of cytosol at 100% degrees C for 1 min before addition to the assay system completely abolished methylation of arsenite. Production of methylarsonate increased in proportion to the arsenite concentration in the assay system; however, 50 microM arsenite inhibited production of dimethylarsinate. Methylarsonate production from carrier-free [73-As]arsenite was not dependent on addition of exogenous S-adenosylmethionine to the assay system. Addition of 0.1 mM S-adenosylmethionine maximized dimethylarsinate production. Addition of 0.1 or 1.0 mM S-adenosylhomocysteine decreased methylation of arsenite, especially dimethylarsinate production. Omission of glutathione from the assay system nearly abolished the methylation of arsenite. Addition of exogenous glutathione to the assay system (up to 20 mM) decreased protein binding of arsenic and increased the production of methylarsonate and dimethylarsinate. The effects of sodium selenite, mercuric chloride, EDTA, p-anisic acid and 2,3-dichloro-alpha-methylbenzylamine on the methylation of arsenite were determined. Addition of 10 microM selenite to the assay system nearly abolished the formation of either methylated species. Addition of 1 or 10 microM mercuric chloride inhibited dimethylarsinate production in a concentration-dependent manner but had little effect on methylarsonate yield. Addition of 10 mM EDTA to the assay system inhibited formation of both methylated metabolites, suggesting that an endogenous divalent cation might be involved in enzymatic methylation of arsenic. Neither p-anisic acid, an inhibitor of cytosolic methyltransferases, nor 2,3-dichloro-alpha-methylbenzylamine, an inhibitor of microsomal methyltransferases, inhibited the conversion of inorganic arsenic to mono- or dimethylated metabolites.

Magnetic resonance microscopy--a new tool for the toxicologic pathologist.

Parallel to its many applications in medical imaging, magnetic resonance (MR) microscopy is a potentially powerful tool in toxicologic pathology. Because of the intrinsic qualities of MR microscopy (noninvasiveness, 3-dimensionality, and slicing in any chosen plane), the scientist has a new means by which to investigate different types of lesions based on differential contrast. By choosing appropriate proton stains to probe the state of the water in tissues, organ structure and vasculature can be seen and progressive lesion development can be followed in a given animal. This paper discusses toxicologic pathology applications for MR microscopy and compares MR microscopy with conventional histopathology using a time-course study of bromobenzene-induced hepatotoxicity in rats. Hematoxylin and eosin (H&E)-stained histological sections are compared with MR microscopy images from fixed tissue blocks to demonstrate one of the applications of MR microscopy to toxicologic pathology. The results indicate that MR microscopy is as sensitive as conventional H&E staining in detecting bromobenzene-induced hepatic lesions.

Time dependence of accumulation and binding of inorganic and organic arsenic species in rabbit erythrocytes.

The uptake by rabbit erythrocytes of 0.4 mM arsenate, As(V), monomethylarsinate, MMA(V) and dimethylarsonate, DMA(V) were compared over 24 h. In membrane-free hemolysate, the distribution of As between proteins (10 kDa) and ultrafiltrate was determined by ultrafiltration and arsenic species in the ultrafiltrate were identified by thin layer chromatography methods. 1H spin-echo Fourier transform NMR was used to follow the binding of these arsenic species to glutathione (GSH). 31P-NMR was used to observe their effects on high-energy adenine nucleotide levels (ATP, ADP). These results demonstrate that As(III) readily accumulates in cells, reaches a quasi-plateau at 78% of the total As in the incubation after 1 h and 88% of the total As after 24 h. On average, 20% of the total erythrocyte As(III) burden is associated with the protein fraction, particularly with hemoglobin (Hb). About 68% of the erythrocyte As(III) burden is bound to GSH. As(III) has no effect on ATP levels during a 5-h incubation. By comparison, As(V) enters erythrocytes more slowly (53% of the total As after 5 h). Erythrocytes take up 81% of the As(V) in the reaction system after a 24 h incubation. Of the total As burden in As(V)-exposed erythrocytes, 22% was associated with the proteins (10 kDa) and possibly reduced to As(III) and 59% was in the ultrafiltrate (8% as As(III) and 51% as As(V)). This finding indicates that, over a 24 h incubation period, the reduction of As(V) to As(III) may account for 30% of the total As in rabbit erythrocytes. As(V) present in the erythrocytes enters the phosphate pool and depletes ATP. In comparison, about 65% of the total MMA(V) or about 44% of the total DMA(V) in the incubation system is taken up by rabbit erythrocytes during a 24 h incubation. Neither organoAs species perturbed the Hb signals observed by spin-echo Fourier transform NMR and the binding to GSH was minimal. Unlike As(V), MMA(V) and DMA(V) do not perturb phosphate metabolism, showing that, despite their pentavalent oxidation state, these arsenic species are not analogs for phosphate.

Identification of methylated metabolites of inorganic arsenic by thin-layer chromatography.

TLC on cellulose plates was used to identify methylated products of inorganic arsenic metabolism (monomethylarsonate and dimethylarsinate) in biological samples. Two solvent systems were tested: methanol-ammonium hydroxide (8:2) and isopropanol-acetic acid-water (10:1:2.5). The latter solvent system produced the most satisfactory separation of radiolabelled methylated arsenic compounds in aqueous solution, in rat liver cytosol incubated with carrier-free or 1 microM [73As]arsenite and in urine of mice given carrier-free [73As]arsenate or 5 mg of [73As]arsenate/kg per os. Oxidation of samples by hydrogen peroxide improved the separation and quantitation of monomethylarsonate in both biological matrices.

Complexation of arsenic species in rabbit erythrocytes.

The binding of arsenite, As(III), and arsenate, As(V), by molecules in the intracellular compartment of rabbit erythrocytes has been studied by 1H- and 31P-NMR spectroscopy, uptake of 73As, and ultrafiltration experiments. For intact erythrocytes to which 0.1-0.4 mM arsenite was added, direct evidence was obtained for entry of 76% within 1/2 h and subsequent binding of As(III) by intracellular glutathione and induced changes in the hemoglobin structure (NMR), likely due to binding of As(III). These results were compared with the effect of addition of As(V) on intact erythrocytes and revealed that a smaller amount of As(V) (approximately 25%) enters the cells; the main fraction of As(V) enters the phosphate pathway, depletes ATP, and increases Pi. In contrast, As(III) did not affect the ATP level. Both 1H- and 31P-NMR data indicated striking differences between As(III) and As(V) behavior when incubated with rabbit erythrocytes. These differences were confirmed by 73As uptake and binding experiments. meso-2,3-Dimercaptosuccinic acid (DMSA), a dithiol ligand, released glutathione from its arsenite complexes in erythrocytes.

Reduction and binding of arsenate and dimethylarsinate by glutathione: a magnetic resonance study.

By observing the chemical shifts of the proton and carbon-13 nuclei of reduced glutathione, the interactions of arsenate, arsenite and dimethylarsinate with this tripeptide have been characterized. These spectral studies show the reduction and complexation of arsenic to be a two-step process. Initially, the oxidation of 2 mol of glutathione reduces arsenate to arsenite. Then, 3 mol of glutathione are consumed in the formation of a glutathione-arsenite complex. Similar experiments with arsenite identified a (glutathione)3-arsenite complex; however, no oxidized glutathione was detected. The arsenite binding site in the glutathione-arsenite complex is the cysteinyl sulfhydryl. The glutathione-arsenite complex is stable over the pH range from 1.5 to 7.0-7.5. At higher pH, dissociation occurs releasing reduced glutathione. For a glutathione to dimethylarsinate ratio of 3, oxidized glutathione is also coupled with a reduction to trivalent dimethylarsinous acid, prior to the formation of a 1:1 glutathione-dimethylarsinite complex. The role of reduced glutathione in the metabolism of arsenic is consistent with the previously described effects of this agent on the organismic toxicity of arsenic.

Transfer of arsenite from glutathione to dithiols: a model of interaction.

The interactions of arsenate and arsenite with meso-2,3-dimercaptosuccinic acid (DMSA) have been characterized using carbon-13 nuclear magnetic resonance. These studies show that DMSA reduces arsenate to arsenite and complexes arsenite. Monitoring the carbon-13 signals of complexed DMSA and liberated glutathione shows that DMSA readily extracts arsenite from a (glutathione)3-arsenite complex, proving the affinity of arsenite for dithiols is greater than that for monothiols. Competition between DMSA (vicinal thiols) and dithioerythritol (1,4-dimercapto-2,3-butanediol) for binding of arsenite indicates that the binding affinity is inversely related to the distance between the two thiol groups. On the basis of these findings, a model for the interaction of arsenic with mono- and dithiol-containing molecules is proposed.

Interaction of inorganic mercury salts with model and red cell membranes: importance of lipid binding sites.

The effect of two mercury salts, HgCl2 and Hg(NO3)2, on the thermotropic properties of phosphatidylserine (PS) model membranes and sonicated rat red cell membranes was investigated by fluorescence polarization. Both Hg(II) salts abolished the phase transition and decreased the membrane fluidity by interacting with PS. Maximal effect was observed at a Hg/PS ratio of 2.5-5 for mercuric chloride and at 1.5 for the nitrate salt. For both mercury compounds, 10 mM NaCl protected model membranes from the effects of Hg(II). HgCl2 and Hg(NO3)2 also decreased the fluidity of rat red cell membranes. Maximal effect was observed for 0.4 mM HgCl2 and 0.6 mM Hg(NO3)2, with 0.0125 mg protein/ml. Addition of NaCl to the Hg(II)-red cell system decreased the Hg(II)-induced perturbation of the thermotropic properties. For both membrane systems, the effects observed with Hg(NO3)2 were greater than those with HgCl2, which can be accounted for by the absence of competition with chloride ions in samples containing Hg(NO3)2.[Cl-] governs the availability of Hg(II) by determining its chemical speciation: increasing [Cl-] generates HgCl3- and HgCl4(2-), which do not interact with lipid binding sites. These results indicate that besides protein thiol groups, Hg(II)-lipid binding sites play an important role in the interaction of Hg(II) with red cell membranes that is qualitatively different from Hg(II) binding to protein thiol groups.

Specific interactions of mercury chloride with membranes and other ligands as revealed by mercury-NMR.

High resolution mercury nuclear magnetic resonance (199Hg-NMR) experiments have been performed in order to monitor mercury chemical speciation when HgCl2 is added to water solutions and follow mercury binding properties towards biomembranes or other ligands. Variations of 199Hg chemical shifts by several hundred ppm depending upon pH and/or pCl changes or upon ligand or membrane addition afforded to determine the thermodynamic parameters which describe the equilibria between the various species in solution. By comparison to an external reference, the decrease in concentration of mercury species in solution allowed to estimate the amount as well as the thermodynamic parameters of unlabile mercury-ligand or mercury-membrane complexes. Hence, some buffer molecules can be classified in a scale of increasing complexing power towards Hg(II): EGTA greater than Tris greater than HEPES. In contrast, MOPS, Borax, phosphates and acetates show little complexation properties for mercury, in our experimental conditions. Evidence for complexation with phosphatidylethanolamine (PE), phosphatidylserine (PS) and human erythrocyte membranes has been found. Hg(II) does not form complexes with egg phosphatidylcholine membranes. Interaction with PE and PS model membranes can be described by the presence of two mercury sites, one labile, the other unlabile, in the NMR time scale. In the labile site Hg(PE) and Hg(PS)2 would be formed whereas in the unlabile site Hg(II) would establish bridges between three PE or PS molecules. Calculated thermodynamic data clearly indicate that PE is a better complexing agent than PS. Evidence is also found that complexation with lipids uses at first the HgCl2 species. Interestingly, mercury complexation with ligands or membranes can be completely reversed by addition of decimolar NaCl solutions. Minute mechanisms for mercury complexation with the primary amine of PE or PS membrane head groups are discussed.