Whole genome transcriptomics reveals global effects including up-regulation of Francisella pathogenicity island gene expression during active stringent response in the highly virulent Francisella tularensis subsp. tularensis SCHU S4.

During conditions of nutrient limitation bacteria undergo a series of global gene expression changes to survive conditions of amino acid and fatty acid starvation. Rapid reallocation of cellular resources is brought about by gene expression changes coordinated by the signalling nucleotides' guanosine tetraphosphate or pentaphosphate, collectively termed (p)ppGpp and is known as the stringent response. The stringent response has been implicated in bacterial virulence, with elevated (p)ppGpp levels being associated with increased virulence gene expression. This has been observed in the highly pathogenic Francisella tularensis sub spp. tularensis SCHU S4, the causative agent of tularaemia. Here, we aimed to artificially induce the stringent response by culturing F. tularensis in the presence of the amino acid analogue l-serine hydroxamate. Serine hydroxamate competitively inhibits tRNAser aminoacylation, causing an accumulation of uncharged tRNA. The uncharged tRNA enters the A site on the translating bacterial ribosome and causes ribosome stalling, in turn stimulating the production of (p)ppGpp and activation of the stringent response. Using the essential virulence gene iglC, which is encoded on the Francisella pathogenicity island (FPI) as a marker of active stringent response, we optimized the culture conditions required for the investigation of virulence gene expression under conditions of nutrient limitation. We subsequently used whole genome RNA-seq to show how F. tularensis alters gene expression on a global scale during active stringent response. Key findings included up-regulation of genes involved in virulence, stress responses and metabolism, and down-regulation of genes involved in metabolite transport and cell division. F. tularensis is a highly virulent intracellular pathogen capable of causing debilitating or fatal disease at extremely low infectious doses. However, virulence mechanisms are still poorly understood. The stringent response is widely recognized as a diverse and complex bacterial stress response implicated in virulence. This work describes the global gene expression profile of F. tularensis SCHU S4 under active stringent response for the first time. Herein we provide evidence for an association of active stringent response with FPI virulence gene expression. Our results further the understanding of the molecular basis of virulence and regulation thereof in F. tularensis. These results also support research into genes involved in (p)ppGpp production and polyphosphate biosynthesis and their applicability as targets for novel antimicrobials.

Engineering of high yield production of L-serine in Escherichia coli.

L-serine is a widely used amino acid that has been proposed as a potential building block biochemical. The high theoretical yield from glucose makes a fermentation based production attractive. In order to achieve this goal, serine degradation to pyruvate and glycine in E. coli MG1655 was prevented by deletion of three L-serine deaminases sdaA, sdaB, and tdcG, as well as serine hydroxyl methyl transferase (SHMT) encoded by glyA. Upon overexpression of the serine production pathway, consisting of a feedback resistant version of serA along with serB and serC, this quadruple deletion strain showed a very high serine production yield (0.45 g/g glucose) during small-scale batch fermentation in minimal medium. Serine, however, was found to be highly toxic even at low concentrations to this strain, which lead to slow growth and production during fed batch fermentation, resulting in a serine production of 8.3 g/L. The production strain was therefore evolved by random mutagenesis to achieve increased tolerance towards serine. Additionally, overexpression of eamA, a cysteine/homoserine transporter was demonstrated to increase serine tolerance from 1.6 g/L to 25 g/L. During fed batch fermentation, the resulting strain lead to the serine production titer of 11.7 g/L with yield of 0.43 g/g glucose, which is the highest yield reported so far for any organism.

Synthesis of pyrazole peptidomimetics and their inhibition against A549 lung cancer cells.

A series of novel pyrazole peptidomimetics was synthesized from 3-aryl-1-arylmethyl-1H-pyrazole-5-carboxylic acid and amino acid ester. Structures of the compounds were characterized by means of IR, (1)H NMR and mass spectroscopy. Compounds 5e and 5k suppress effectively the growth of A549 lung cancer cells. Preliminary research on the mechanism of action showed that the inhibition might perform through combination of apoptosis, autophagy and cell cycle arrest.

Effects of non-steroidal antiinflammatory drugs on D-serine-induced oxidative stress in vitro.

Inflammation is deleterious for organs with reduced capacity of regeneration, such as the brain. Recently, studies have focused on investigating the therapeutic effects of nonsteroidal anti-inflammatory drugs (NSAIDs) in Alzheimer's disease, Parkinson's disease, Huntington's disease, and multiple sclerosis. Excitotoxicity is the pathological process when receptors for the excitatory neurotransmitter glutamate, such as the N-methyl-D-aspartate (NMDA), receptors are overactivated. This process may be involved in neurodegenerative diseases. D-serine is one of the coagonist of NMDA receptors, and increased levels of D-serine are associated with excitotoxicity. In our study, the potential neuroprotective effects of mefenamic acid, acetaminophen, and naproxen sodium were investigated against D-serine-induced oxidative stress in the rat brain in vitro. To show their potential neuroprotective properties, NSAIDs were incubated with D-serine and reactive oxygen species (ROS), malondialdehyde, and protein carbonyl content of the brain after different treatments were measured. Our results demostrate that NSAIDs used in the present study significantly reduced ROS production, lipid peroxidation, and protein oxidation against D-serine treatment.

D-serine treatment induces oxidative stress in rat brain.

D-serine plays a significant role in neuronal activity, including learning, memory, neuronal migration at developmental stages, and cell-death signaling. It has been also suggested that D-serine can potantiate the neurotoxicity induced by N-methyl-D-aspartate (NMDA) receptor activation due to its coagonist function. However, little is known about the role of D-serine in oxidative stress mechanisms. The aim of this study was to determine the possible neurotoxic or oxidative effects of the dose- (50-200 mg/kg) and time-dependent (2 or 6 hours) D-serine administration on lipid, protein, DNA, mitochondrial integrity (i.e., function), levels of antioxidant enzyme activities (e.g., catalase, glutathione peroxidase, and superoxide dismutase), and glutathione (GSH) in the rat brain. Our results showed that D-serine significantly increases the levels of lipid peroxidation, protein carbonyls, and DNA damage. In addition, D-serine treatment changes cellular antioxidant status due to the decreased levels of antioxidant enzymes, GSH, and mitochondrial function. Therefore, it is concluded that the regulation of D-serine levels in the brain may be an important target for the development of neuroprotective strategies against neurodegenerative processes where excitotoxicity is involved.

Synthesis of actin-depolymerizing compounds.

The artificial actin-depolymerizing compounds 3-6, based on aplyronine A, an actin-depolymerizing antitumor marine macrolide, were synthesized, and their actin-depolymerizing activities and cytotoxicities were evaluated.

Pharmacokinetics and toxicokinetics of d-serine in rats.

In the mammalian brain, d-serine acts as a co-agonist at the glycine-binding site on the N-methyl-d-aspartate receptor. Because plasma d-serine levels are significantly lower in patients with schizophrenia than in healthy subjects, d-serine has been proposed as a potential therapeutic agent for schizophrenia treatment. However, d-serine has a nephrotoxic effect in rats at high doses. The purpose of this study was to investigate the relationship between the plasma kinetics of d-serine and nephrotoxicity in rats. We administered d-serine intravenously (iv), orally (po), or intraperitoneally (ip) to male Wistar rats, and performed gas chromatography-mass spectrometry to measure the plasma concentrations of d- and l-serine. After iv administration (0.1 mmol/kg body weight (bw)), plasma d-serine declined multiexponentially with an elimination t1/2 of 108 ± 16 min, and the total clearance was 7.9 ± 0.9 ml/min/kg bw. The oral bioavailability of d-serine was estimated to be 94 ± 27%. To evaluate the dose-response relationship of d-serine-induced kidney injury and the plasma kinetics of d-serine, we injected d-serine into rats ip in doses ranging from 0.6 to 4.8 mmol/kg bw. Twenty-four hours after d-serine administration, histological changes indicating renal damage were observed in the kidneys of rats who received d-serine at doses of 1.8-4.8 mmol/kg bw; the severity of the tubular injury increased with increasing d-serine dose. When the Cmax value of d-serine was approximately >2 µmol/ml, the plasma creatinine increased remarkably 24 h after d-serine administration. This suggests that the Cmax of d-serine could be a good predictor of d-serine-induced nephrotoxicity.

D-Serine exposure resulted in gene expression changes indicative of activation of fibrogenic pathways and down-regulation of energy metabolism and oxidative stress response.

Renal toxicity can commonly occur after exposure to xenobiotics, pharmaceutical agents or environmental pollutants. Changes in the gene expression in kidney parenchymal cells that precede and/or accompany renal injury may be hallmark critical events in the onset of pathologic changes of renal functions. Over the last several years, transcriptomic analysis has evolved to enable simultaneous analysis of the expression profiles of tens of thousands of genes in response to various endogenous and exogenous stimuli. In this study, we investigated gene expression changes in the kidney after acute exposure to a nephrotoxin, D-serine, which targets the proximal tubule of the kidney. Male F-344 rats injected intraperitoneally with a single dose of D-serine (5, 20, 50, 200 or 500 mg/kg), and gene expression profiles in the kidney were determined using the Affymetrix RAE230A gene arrays at 96 h post-dosing. D-serine treatment resulted in the up- and down-regulation of 1158 and 749 genes, respectively, over the entire dose range based on the intersection of the results of t-test, p<0.01 over two consecutive doses, and ANOVA with Bonferonni correction for multiple testing. Interestingly, both the up-and down-regulated genes show a unified dose response pattern as revealed in the self-organized map clustering analysis using the expression profiles of the 1907 differentially expressed genes as input data. There appears to be minimal changes in the expression level of these genes in the dose range of 5-50 mg/kg, while the most prominent changes were observed at the highest doses tested, i.e. 200 and 500 mg/kg. Pathway analysis of the differentially expressed genes showed perturbation of a large number of biological processes/pathways after d-serine exposure. Among the up-regulated pathways are actin cytoskeleton biogenesis and organization, apoptosis, cell cycle regulation, chromatin assembly, excision repair of damaged DNA, DNA replication and packaging, protein biosynthesis, metabolism and transport, inflammatory response, proteasome-mediated degradation of oxidatively damaged cytosolic proteins, Ras protein signal transduction, TGF-beta signaling pathway and mRNA transcription, processing, splicing and transport. On the other hand, major metabolic pathways, which include carbohydrate metabolism, TCA cycle, oxidative phosphorylation, ATP synthesis coupled electron transport, amino acid metabolism and transport, lipid metabolism, nucleotide metabolism, and vitamin metabolism, and oxidative stress response including induction of antioxidant genes and glutathione metabolism are down-regulated. As tubular epithelia have strong energy demand for normal functions, down-regulation of energy metabolism after D-serine treatment may be related to the mechanism of its nephrotoxicity. In addition, hydrogen peroxide, a reactive oxygen species, is produced as a byproduct of the metabolism of D-serine by D-amino acid oxidase in the peroxisomes of the tubular epithelia. Down-regulation of pathways for antioxidant genes induction and glutathione metabolism will likely exacerbate the cytotoxicity of this reactive oxygen species. The observation that the genes involved in apoptosis, DNA repair, proteasome pathway for the degradation of oxidatively damaged cytosolic proteins were up-regulated lends some supports to this premise. Up-regulation of pathways of cell proliferation cycle, DNA replication and gene expression process, including mRNA transcription, processing, splicing, transport, translation initiation, and protein transport along with protein complex assembly, suggests ongoing tissue repair and regeneration. Consistent with the fibrogenic function of the TGF-beta signaling pathway in various experimental renal diseases, genes encoding major extracellular matrix components such as collagens, laminins, fibronectin 1 and tenascins are also strongly up-regulated. Taken together, the results of this study provide important insights into the molecular mechanism of D-serine nephrotoxicity, as well as the activation of specific cellular pathways in response to this toxic insult.

Evaluation of oxidative stress in D-serine induced nephrotoxicity.

It has been suggested that oxidative stress is involved in d-serine-induced nephrotoxicity. The purpose of this study was to assess if oxidative stress is involved in this experimental model using several approaches including (a) the determination of several markers of oxidative stress and the activity of some antioxidant enzymes in kidney and (b) the use of compounds with antioxidant or prooxidant effects. Rats were sacrificed at several periods of time (from 3 to 24h) after a single i.p. injection of d-serine (400mg/kg). Control rats were injected with l-serine (400mg/kg) and sacrificed 24h after. The following markers were used to assess the temporal aspects of renal damage: (a) urea nitrogen (BUN) and creatinine in blood serum, (b) kidney injury molecule (KIM-1) mRNA levels, and (c) tubular necrotic damage. In addition, creatinine clearance, proteinuria, and urinary excretion of N-acetyl-beta-d-glucosaminidase (NAG) were measured 24h after d-serine injection. Protein carbonyl content, malondialdehyde (MDA), 4-hydroxy-2-nonenal (4-HNE), fluorescent products of lipid peroxidation, reactive oxygen species (ROS), glutathione (GSH) content, and heme oxygenase-1 (HO-1) expression were measured as markers of oxidative stress in the kidney. Additional experiments were performed using the following compounds with antioxidant or pro-oxidant effects before d-serine injection: (a) alpha-phenyl-tert-butyl-nitrone (PBN), a spin trapping agent; (b) 5,10,15,20-tetrakis (4-sulfonatophenyl) porphyrinato iron(III) (FeTPPS), a soluble complex able to metabolize peroxynitrite; (c) aminotriazole (ATZ), a catalase (CAT) inhibitor; (d) stannous chloride (SnCl(2)), an HO-1 inductor; (e) tin mesoporphyrin (SnMP), an HO inhibitor. In the time-course study, serum creatinine and BUN increased significantly on 15-24 and 20-24h, respectively, and KIM-1 mRNA levels increased significantly on 6-24h. Histological analyses revealed tubular necrosis at 12h. The activity of antioxidant enzymes catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase remained unchanged at all times studied. Protein carbonyl content, MDA, 4-HNE, and ROS remained unchanged at all time-points studied. GSH content decreased transiently on 9 and 12h. Interestingly, fluorescent products of lipid peroxidation decreased significantly on 3-24h. HO-1 expression was undetectable by Western blot and the immunohistochemistry studies revealed that the intensity of HO-1 staining was weak. The administration of PBN, FeTPPS, ATZ, SnCl(2), and SnMP did not prevent or enhance renal damage induced by d-serine. Our data taken as a whole suggest that oxidative stress is not involved in the early phase of the nephrotoxicity induced by d-serine.

D-serine is the dominant endogenous coagonist for NMDA receptor neurotoxicity in organotypic hippocampal slices.

D-serine occurs at high levels in the brain, where it is an endogenous coagonist at the "glycine site" of NMDA receptors. However, D-serine action has not been previously compared with that of endogenous glycine, and the relative importance of the two coagonists remains unclear. We now investigated the efficiencies of the two coagonists in mediating NMDA receptor neurotoxicity in organotypic hippocampal slices. Removal of endogenous D-serine from slices was achieved by pretreating the tissue with recombinant D-serine deaminase enzyme. This enzyme is several orders of magnitude more efficient than previous methods to remove D-serine. We report that complete removal of D-serine virtually abolished NMDA-elicited neurotoxicity but did not protect against kainate. Although levels of glycine were 10-fold higher than D-serine, endogenous glycine was ineffective in mediating NMDA receptor neurotoxicity. The effect of endogenous glycine could be observed only after simultaneous removal of endogenous D-serine and blockage of the glycine transporter GlyT1. Our data indicate that D-serine is the dominant coagonist for NMDA receptor-elicited neurotoxicity, mediating all cell death elicited by NMDA in organotypic slices. The results suggest an essential role for this unusual D-amino acid, with implications for the mechanism of neuronal death in the nervous system.

Impact of the gliotoxin L-serine-O-sulphate on cellular metabolism in cultured rat astrocytes.

L-serine-O-sulphate is a member of a group of amino acids collectively called gliotoxins and is a substrate for the high affinity sodium-dependent glutamate transporters. Previous studies have shown that it is toxic to primary cultures of astrocytes but the mode of toxicity is unknown. The current study demonstrates that L-serine-O-sulphate, at a sub-toxic concentration (400 microM), causes significant disruption to glucose and alanine metabolism in cultures of rat cortical astrocytes. More specifically, using (13)C NMR spectroscopy a significant reduction in labelled end products from [1-(13)C]glucose and [3-(13)C]alanine was found in the presence of L-serine-O-sulphate. Additionally, using [2-(13)C]glycine a 27% reduction in de novo glutathione synthesis was observed in the presence of the gliotoxin. Incubation of the cells with L-serine-O-sulphate reduced the activity of alanine and aspartate aminotransferase by 53% and 67%, respectively. Collectively these results show that the gliotoxin, L-serine-O-sulphate, causes major disruptions to metabolic pathways in primary cultures of astrocytes.

Cytotoxicity and sister chromatid exchanges induced in vitro by six anticancer drugs developed in the People's Republic of China.

Growth inhibition in the Chinese hamster cell line V79 and in the human lymphoid cell line Raji and induction of sister chromatid exchange(s) (SCE) in V79 cells after treatment with six anticancer drugs [harringtonine (HRT), homoharringtonine (HHRT), camptothecin (CPT), hydroxycamptothecin (HCPT), lycobetaine (LBT), and oxalysine (OXL)] developed in the People's Republic of China were studied. OXL is a new antibiotic; all other drugs are plant extracts. All drugs caused a dose-dependent growth inhibition in both cell types, as evidenced by decreases in plating efficiencies of V79 cells and in viable cell counts of Raji. However, the degree of inhibition differed widely among the drugs. HRT, HHRT, CPT, and HCPT were the most potent growth inhibitors, LBT was next, and OXL was the least effective inhibitor. SCE analyses were made in V79 cells treated with a drug in the presence or absence of the metabolic activation system S9 mixture (S9 mix), except for the HRT assay in which the S9 mix was not used. CPT, HCPT, and LBT induced a dose-dependent increase in SCE frequencies, while HRT, HHRT, and OXL caused no SCE induction at any dose level used. CPT was the most powerful SCE inducer. HCPT induced SCE but at a much reduced rate when compared to that of CPT. LBT was a weak SCE inducer; SCE induction was seen only in cultures treated with 40 micrograms or more LBT/ml. Addition of the S9 mix did not alter SCE frequencies, indicating that the drugs were direct-acting agents. HRT and HHRT were highly toxic, but they induced no increases in SCE frequency, indicating that cytotoxicity of a compound does not necessarily correlate with SCE induction.

DL-Serine: promoting activity on renal tumorigenesis by N-ethyl-N-hydroxyethylnitrosamine in rats.

Subcutaneous injection of DL-serine increased the number and size of renal tubular cell tumors in male W rats treated with 500 or 1,000 ppm N-ethyl-N-hydroxyethylnitrosamine [(EHEN) CAS: 13147-25-6, 2-(ethylnitrosamino)ethanol]. At the end of the 32-week experiment, the incidences of renal tumors were 95% in rats treated with 1,000 ppm EHEN for 2 weeks and then given three sc injections of DL-serine every 2 weeks, 33% in rats treated with 1,000 ppm EHEN for 2 weeks, and 28% in rats treated with 500 ppm EHEN for 2 weeks and then given three sc injections of DL-serine every 2 weeks. No renal tumors were found in rats treated with 500 ppm EHEN alone or given three sc injections of DL-serine alone every 2 weeks.

D-Serine-induced nephrotoxicity: a HPLC-TOF/MS-based metabonomics approach.

HPLC-MS-based metabonomic analysis was used to investigate urinary metabolic perturbations associated with D-serine-induced nephrotoxicity. D-Serine causes selective necrosis of the proximal straight tubules in the rat kidney accompanied by aminoaciduria, proteinuria and glucosuria. Alderely Park (Wistar-derived) rats were dosed with either D-serine (250 mg/kg ip) or vehicle (deionised water) and urine was collected at 0-12, 12-24, 24-36 and 36-48 h post-dosing. Samples were analysed using a Waters Alliance HT 2795 HPLC system coupled to a Waters Micromass Q-ToF-micro equipped with an electrospray source operating in either positive or negative ion mode. Changes to the urinary profile were detected at all time points compared to control. In negative ion mode, increases were observed in serine (m/z=103.0077), m/z=104.0376 (proposed to be hydroxypyruvate) and glycerate (m/z=105.0215), the latter being metabolites of D-serine. Furthermore, an increase in tryptophan, phenylalanine and lactate and decreases in methylsuccinic acid and sebacic acid were observed. Positive ion analysis revealed a decrease in xanthurenic acid, which has previously been assigned and reported using HPLC-MS following exposure to mercuric chloride and cyclosporine A. A general aminoaciduria, including proline, methionine, leucine, tyrosine and valine was also observed as well as an increase in acetyl carnitine. Investigation of additional metabolites altered as a result of exposure to D-serine is on-going. Thus, HPLC-MS-based metabonomic analysis has provided information concerning the mechanism of D-serine-induced renal injury.

Sodium benzoate attenuates D-serine induced nephrotoxicity in the rat.

D-Serine causes selective necrosis to the straight portion of the rat renal proximal tubules. The onset is rapid, occurring within 3-4 h and accompanied by proteinuria, glucosuria and aminoaciduria. The metabolism of D-serine by D-amino acid oxidase (D-AAO) may be involved in the mechanism of toxicity. D-AAO is localized within the peroxisomes of renal tubular epithelial cells, which is also the location of D-serine reabsorption. To address the role of D-AAO in D-serine-induced nephrotoxicity, we have examined the effect of sodium benzoate (SB) on the renal injury. SB has been shown to be a potent, competitive inhibitor of kidney D-AAO in vitro. Male Alderley Park rats were exposed to D-serine (500 mg/kg i.p.) 1 h after exposure to SB (125, 250, 500 or 750 mg/kg i.p.). Urine was collected for 0-6 h, then terminal plasma samples and kidneys were taken at 6.5 h. A second group of animals was given SB (500 mg/kg) followed by D-serine (500 mg/kg i.p.; 1 h later) and urine was collected after 0-6, 6-24 and 24-48 h. Terminal plasma samples and kidneys were taken at 48 h. 1H NMR spectroscopic analysis of urine, combined with principal component analysis, demonstrated that SB was able to prevent D-serine-induced perturbations to the urinary profile in a dose dependent manner. This was confirmed by measurement of plasma creatinine and urinary glucose and protein and histopathological examination of the kidneys. Assessment 48 h after D-serine administration revealed that nephrotoxicity was observed in animals pre-treated with SB (500 mg/kg) although the extent of injury was less pronounced than following D-serine alone. These results demonstrate that whilst prior exposure to SB prevents the initial onset of D-serine-induced nephrotoxicity, renal injury is still apparent at later time points. D-AAO activity in the kidney was decreased by 50% 1 h after dosing with SB suggesting that inhibition of this enzyme may be responsible for the observed protection.

Novel serine-based gemini surfactants as chemical permeation enhancers of local anesthetics: A comprehensive study on structure-activity relationships, molecular dynamics and dermal delivery.

This work aims at studying the efficacy of a series of novel biocompatible, serine-based surfactants as chemical permeation enhancers for two different local anesthetics, tetracaine and ropivacaine, combining an experimental and computational approach. The surfactants consist of gemini molecules structurally related, but with variations in headgroup charge (nonionic vs. cationic) and in the hydrocarbon chain lengths (main and spacer chains). In vitro permeation and molecular dynamics studies combined with cytotoxicity profiles were performed to investigate the permeation of both drugs, probe skin integrity, and rationalize the interactions at molecular level. Results show that these enhancers do not have significant deleterious effects on the skin structure and do not cause relevant changes on cell viability. Permeation across the skin is clearly improved using some of the selected serine-based gemini surfactants, namely the cationic ones with long alkyl chains and shorter spacer. This is noteworthy in the case of ropivacaine hydrochloride, which is not easily administered through the stratum corneum. Molecular dynamics results provide a mechanistic view of the surfactant action on lipid membranes that essentially corroborate the experimental observations. Overall, this study suggests the viability of these serine-based surfactants as suitable and promising delivery agents in pharmaceutical formulations.

A correlation between a proteomic evaluation and conventional measurements in the assessment of renal proximal tubular toxicity.

4-Aminophenol (4-AP), D-serine, and cisplatin are established rodent nephrotoxins that damage proximal tubules within the renal cortex. Using high throughput 2D gel proteomics to profile protein changes in the plasma of compound-treated animals, we identified several markers of kidney toxicity. Male F344 and Alpk rats were treated with increasing doses of 4-AP, D-serine, or cisplatin, and plasma samples were collected over time. Control groups received saline or nontoxic isomers, L-serine, and transplatin. Plasma proteins that displayed dose- and temporal-dependent regulation in each study were further characterized by mass spectrometry to elucidate the protein identity. Several isoforms of the rat-specific T-kininogen protein were identified in each study. T-kininogen was elevated in the plasma of 4-AP-, D-serine-, and cisplatin-treated animals at early time points, returning to baseline levels 3 weeks after treatment. The protein was not elevated in the plasma of control animals or those treated with nontoxic compounds. We propose that T-kininogen may be required to counteract apoptosis in proximal tubular cells in order to minimize tissue damage following a toxic insult. In addition, T-kininogen may be required to stimulate localized inflammation to aid tissue repair. We also identified several isoforms of the inter-alpha inhibitor H4P heavy chain in the 4-AP and D-serine studies. In each case, the protein expression levels in the blood samples paralleled the extent of kidney toxicity, highlighting the correlation between protein alterations and clinical chemistry endpoints. A further set of proteins correlating with kidney damage was found to be a component of the complement cascade and other blood clotting factors, indicating a contribution of the immune system to the observed toxicity. These observations underscore the value of proteomics in identifying new biomarkers and in the elucidation of mechanisms of toxicity.

D-serine-induced nephrotoxicity: possible interaction with tyrosine metabolism.

D-serine selectively damages renal proximal tubule cells in rats by a mechanism that is not fully understood. Recent proteomic analysis identified that D-serine elevated plasma fumarylacetoacetate hydrolase (FAH). FAH is involved in tyrosine catabolism; hence, this pathway may be involved in mediating the toxicity. This work examines whether 2-(2-nitro-4-trifluoromethylbenzoyl)-cyclohexane-1,3-dione (NTBC), a potent inhibitor of the enzyme 4-hydroxyphenylpyruvate dioxygenase (HPPD) located upstream of FAH, modulates D-serine-induced nephrotoxicity. Rats were pretreated with NTBC (0.5 mg/kg p.o.) or corn oil and then 30 min later given either D-serine (250 mg/kg i.p.) or water. Urine was collected every 12 h until termination (48 h) and analysed by 1H NMR spectroscopy and principal component analysis (PCA). Markers of proximal tubule injury were evident in urine following treatment with D-serine and NTBC + D-serine. PCA could not distinguish between these urine samples suggesting that NTBC does not effect the development of nephrotoxicity. Clinical chemistry analysis of urine and terminal plasma samples and histopathological examination of the kidneys confirmed this. NTBC alone caused a marked increase in the excretion of 4-hydroxyphenylpyruvate (HPPA) and 4-hydroxyphenyllactate (HPLA); however, HPPA and HPLA excretion was minimal following NTBC + D-serine. Instead marked tyrosinuria was observed suggesting that D-serine-induced renal damage markedly affects the handling of increased levels of HPPA and HPLA resulting from the inhibition of HPPD.

Reversible inhibition of the glycine transporter GlyT2 circumvents acute toxicity while preserving efficacy in the treatment of pain.

BACKGROUND AND PURPOSE: Available medications for chronic pain provide only partial relief and often cause unacceptable side effects. There is therefore a need for novel molecular targets to develop new therapeutics with improved efficacy and tolerability. Despite encouraging efficacy data in rodents with inhibitors of the neuronal glycine transporter-2 (GlyT2), there are also some reports of toxicity and their development was discontinued. EXPERIMENTAL APPROACH: In order to clarify the possibility of targeting GlyT2 for the treatment of pain, we have used an integrated approach comprising in vitro pharmacology, selectivity, bioavailability, in vivo efficacy and safety assessment to analyse the properties and efficacy of ALX-1393 and Org-25543, the two published GlyT2 inhibitors from which in vivo data are available. KEY RESULTS: We report that these compounds have a different set of undesirable properties that limit their usefulness as pharmacological tools. Importantly, we discover that inhibitors of GlyT2 can exert an apparent reversible or irreversible inhibition of the transporter and describe a new class of reversible GlyT2 inhibitors that preserves efficacy while avoiding acute toxicity. CONCLUSIONS AND IMPLICATIONS: Our pharmacological comparison of two closely related GlyT2 inhibitors with different modes of inhibition provides important insights into their safety and efficacy profiles, uncovering that in the presence of a GlyT2 mechanism-based toxicity, reversible inhibitors might allow a tolerable balance between efficacy and toxicity. These findings shed light into the drawbacks associated with the early GlyT2 inhibitors and describe a new mechanism that might serve as the starting point for new drug development.

Simultaneous determination of serine enantiomers in plasma using Mosher's reagent and stable isotope dilution gas chromatography-mass spectrometry.

D-Serine is a co-agonist of the N-methyl-D-aspartate receptor in glutamate neurotransmission and has been proposed as a potential therapeutic agent for schizophrenia. However, D-serine also acts as a nephrotoxic substance in rats at high doses. To investigate the pharmacokinetics and toxicokinetics of D-serine, a method for the stereoselective determination of serine enantiomers in rat plasma was developed using GC-MS with selected ion monitoring (GC-MS-SIM). DL-[(2)H(3)]Serine was used as an internal standard to account for losses associated with the extraction, derivatization and chromatography. Serine enantiomers were purified by cation-exchange chromatography using BondElut SCX cartridge and derivatized with HCl in methanol to form methyl ester followed by subsequent N,O-diacylation with optically active (+)-α-methoxy-α-trifluoromethylphenylacetyl chloride to form epimeric amide. Quantitation was performed by SIM of the molecular-related ions of the epimers in the chemical ionization mode. The intra- and inter-day reproducibility of the assay was less than 5% for D-serine and 3% for L-serine. The method was successively applied to study the pharmacokinetics of D-serine in rats.