8-acetoxy-trisulfopyrene as the first activatable fluorogenic probe for add-and-read assessment of Organic anion-transporting polypeptides, OATP1B1, OATP1B3, and OATP2B1.

Organic anion-transporting polypeptides, OATP1B1, OATP1B3, and OATP2B1 are multispecific membrane proteins mediating the hepatocellular uptake of structurally diverse endo- and exogenous compounds, including various kinds of drugs. Co-administration of OATP1B/2B1 substrates may lead to altered pharmacokinetics or even toxicity. Therefore, the study of the interaction with these OATPs is essential in drug development and is recommended by international regulatory agencies, the FDA, EMA, and PMDA. In general, radiolabeled indicators are used to measure drug interactions of OATPs, and, lately, fluorescent probes are also gaining wider application in OATP tests. However, all of the currently available methods (either radioactive or fluorescence-based) comprise multiple steps, including the removal of the indicator in the end of the experiment. Hence, they are not ideally suited for high-throughput screening. In the current study, in order to find an indicator allowing real-time assessment of hepatic OATP function, we searched for an activatable fluorogenic OATP substrate. Here, we show that 8-acetoxypyrene-1,3,6-trisulfonate (Ace), a fluorogenic derivative of the hepatic OATP substrate pyranine (8-hydroxypyrene-1,3,6-trisulfonate) enters the cells via OATP1B1/3 or OATP2B1 function. In living cells, Ace is then converted into highly fluorescent pyranine, allowing "no-wash" measurement of OATP function and drug interactions. Furthermore, we demonstrate that Ace can be used in an indirect assay termed as competitive counterflow suitable to distinguish between transported substrates and inhibitors of OATP1B1. The fluorescence-based methods described here are unique and open the way toward high-throughput screening of interactions between new molecular entities and OATPs.

Computer-Aided Design and Synthesis of (Functionalized quinazoline)-(α-substituted coumarin)-arylsulfonate Conjugates against Chikungunya Virus.

Chikungunya virus (CHIKV) has repeatedly spread via the bite of an infected mosquito and affected more than 100 countries. The disease poses threats to public health and the economy in the infected locations. Many efforts have been devoted to identifying compounds that could inhibit CHIKV. Unfortunately, successful clinical candidates have not been found yet. Computations through the simulating recognition process were performed on complexation of the nsP3 protein of CHIKV with the structures of triply conjugated drug lead candidates. The outcomes provided the aid on rational design of functionalized quinazoline-(α-substituted coumarin)-arylsulfonate compounds to inhibit CHIKV in Vero cells. The molecular docking studies showed a void space around the ß carbon atom of coumarin when a substituent was attached at the α position. The formed vacancy offered a good chance for a Michael addition to take place owing to steric and electronic effects. The best conjugate containing a quinazolinone moiety exhibited potency with EC50 = 6.46 µM, low toxicity with CC50 = 59.7 µM, and the selective index (SI) = 9.24. Furthermore, the corresponding 4-anilinoquinazoline derivative improved the anti-CHIKV potency to EC50 = 3.84 µM, CC50 = 72.3 µM, and SI = 18.8. The conjugate with 4-anilinoquinazoline exhibited stronger binding affinity towards the macro domain than that with quinazolinone via hydrophobic and hydrogen bond interactions.

Host-Guest Complexation of Oxaliplatin and Para-Sulfonatocalix[n]Arenes for Potential Use in Cancer Therapy.

P-sulfonatocalix[n]arenes have demonstrated a great potential for encapsulation of therapeutic drugs via host-guest complexation to improve solubility, stability, and bioavailability of encapsulated drugs. In this work, guest-host complexes of a third-generation anticancer drug (oxaliplatin) and p-4-sulfocalix[n]arenes (n = 4 and 6; p-SC4 and p-SC6, respectively) were prepared and investigated, using 1H NMR, UV, Job's plot analysis, and DFT calculations, for use as cancer therapeutics. The peak amplitude of the prepared host-guest complexes was linearly proportional to the concentration of oxaliplatin in the range of 1.0 × 10-5 M-1 to 2.1 × 10-4 M-1. The reaction stoichiometry between either p-SC4 or p-SC6 and oxaliplatin in the formed complexes was 1:1. The stability constants for the complexes were 5.07 × 104 M-1 and 6.3 × 104 M-1. These correspond to complexation free energy of -6.39 and -6.52 kcal/mol for p-SC4 and p-SC6, respectively. Complexation between oxaliplatin and p-SC4 or p-SC6 was found to involve hydrogen bonds. Both complexes exhibited enhanced biological and high cytotoxic activities against HT-29 colorectal cells and MCF-7 breast adenocarcinoma compared to free oxaliplatin, which warrants further investigation for cancer therapy.

Claudin-18 deficiency is associated with airway epithelial barrier dysfunction and asthma.

BACKGROUND: Epithelial barrier dysfunction and increased permeability may contribute to antigen sensitization and disease progression in asthma. Claudin-18.1 is the only known lung-specific tight junction protein, but its contribution to airway barrier function or asthma is unclear. OBJECTIVES: We sought to test the hypotheses that claudin-18 is a determinant of airway epithelial barrier function that is downregulated by IL-13 and that claudin-18 deficiency results in increased aeroantigen sensitization and airway hyperresponsiveness. METHODS: Claudin-18.1 mRNA levels were measured in airway epithelial brushings from healthy controls and patients with asthma. In patients with asthma, claudin-18 levels were compared with a three-gene-mean marker of TH2 inflammation. Airway epithelial permeability changes due to claudin-18 deficiency were measured in 16HBE cells and claudin-18 null mice. The effect of IL-13 on claudin expression was determined in primary human airway epithelial cells and in mice. Airway hyperresponsiveness and serum IgE levels were compared in claudin-18 null and wild-type mice following aspergillus sensitization. RESULTS: Epithelial brushings from patients with asthma (n = 67) had significantly lower claudin-18 mRNA levels than did those from healthy controls (n = 42). Claudin-18 levels were lowest among TH2-high patients with asthma. Loss of claudin-18 was sufficient to impair epithelial barrier function in 16HBE cells and in mouse airways. IL-13 decreased claudin-18 expression in primary human cells and in mice. Claudin-18 null mice had significantly higher serum IgE levels and increased airway responsiveness following intranasal aspergillus sensitization. CONCLUSIONS: These data support the hypothesis that claudin-18 is an essential contributor to the airway epithelial barrier to aeroantigens. Furthermore, TH2 inflammation suppresses claudin-18 expression, potentially promoting sensitization and airway hyperresponsiveness.

The 5-phosphatase OCRL mediates retrograde transport of the mannose 6-phosphate receptor by regulating a Rac1-cofilin signalling module.

Mutations in the OCRL gene encoding the phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) 5-phosphatase OCRL cause Lowe syndrome (LS), which is characterized by intellectual disability, cataracts and selective proximal tubulopathy. OCRL localizes membrane-bound compartments and is implicated in intracellular transport. Comprehensive analysis of clathrin-mediated endocytosis in fibroblasts of patients with LS did not reveal any difference in trafficking of epidermal growth factor, low density lipoprotein or transferrin, compared with normal fibroblasts. However, LS fibroblasts displayed reduced mannose 6-phosphate receptor (MPR)-mediated re-uptake of the lysosomal enzyme arylsulfatase B. In addition, endosome-to-trans Golgi network (TGN) transport of MPRs was decreased significantly, leading to higher levels of cell surface MPRs and their enrichment in enlarged, retromer-positive endosomes in OCRL-depleted HeLa cells. In line with the higher steady-state concentration of MPRs in the endosomal compartment in equilibrium with the cell surface, anterograde transport of the lysosomal enzyme, cathepsin D was impaired. Wild-type OCRL counteracted accumulation of MPR in endosomes in an activity-dependent manner, suggesting that PI(4,5)P(2) modulates the activity state of proteins regulated by this phosphoinositide. Indeed, we detected an increased amount of the inactive, phosphorylated form of cofilin and lower levels of the active form of PAK3 upon OCRL depletion. Levels of active Rac1 and RhoA were reduced or enhanced, respectively. Overexpression of Rac1 rescued both enhanced levels of phosphorylated cofilin and MPR accumulation in enlarged endosomes. Our data suggest that PI(4,5)P(2) dephosphorylation through OCRL regulates a Rac1-cofilin signalling cascade implicated in MPR trafficking from endosomes to the TGN.

Nanobiosensors exploiting specific interactions between an enzyme and herbicides in atomic force spectroscopy.

The development of sensitive methodologies for detecting agrochemicals has become important in recent years due to the increasingly indiscriminate use of these substances. In this context, nanosensors based on atomic force microscopy (AFM) tips are useful because they provide higher sensitivity with operation at the nanometer scale. In this paper we exploit specific interactions between AFM tips functionalized with the enzyme acetolactate synthase (ALS) to detect the ALS-inhibitor herbicides metsulfuron-methyl and imazaquin. Using atomic force spectroscopy (AFS) we could measure the adhesion force between tip and substrate, which was considerably higher when the ALS-functionalized tip (nanobiosensor) was employed. The increase was approximately 250% and 160% for metsulfuron-methyl and imazaquin, respectively, in comparison to unfunctionalized probes. We estimated the specific enzyme-herbicide force by assuming that the measured force comprises an adhesion force according to the Johnson-Kendall-Roberts (JKR) model, the capillary force and the specific force. We show that the specific, biorecognition force plays a crucial role in the higher sensitivity of the nanobiosensor, thus opening the way for the design of similarly engineered tips for detecting herbicides and other analytes.

Monitoring the active transport of efflux pumps after their reconstitution into proteoliposomes: caveats and keys.

There is an acute need for a functional assay allowing the investigation of efflux pumps. A dedicated procedure was previously developed, but although it was unambiguous, it suffered from a lack of reproducibility. We describe an optimization of the procedure that makes the assay much more robust.

Augmentation of tribenuron methyl removal from polluted soil with Bacillus sp. strain BS2 and indigenous earthworms.

Tribenuron methyl (TBM) is a member of the sulfonylurea herbicide family and is widely used worldwide. In this study, TBM-degrading bacteria were enriched with TBM as potential carbon, nitrogen or sulfur source, and 44 bacterial isolates were obtained. These isolates were phylogenetically diverse, and were grouped into 25 operational taxonomic units and 14 currently known genera. Three representatives, Bacillus sp. strain BS2, Microbacterium sp. strain BS3, and Cellulosimicrobium sp. strain BS11, were selected, and their growth and TBM removal from culture broth were investigated. In addition, indigenous earthworms were collected and applied to augment TBM degradation in lab-scale soil column experiments. Results demonstrated that Bacillus sp. strain BS2 and earthworms significantly increased TBM removal during soil column experiments.

Study of biochemical pathway and enzyme involved in metsulfuron-methyl degradation by Ancylobacter sp. XJ-412-1 isolated from soil.

Ancylobacter sp. XJ-412-1, capable of degrading metsulfuron-methyl, was isolated from sulfonylurea-contaminated soil. When metsulfuron-methyl was provided as the sole carbon source, more than 90.5% of metsulfuron-methyl at concentration of 50 mg l(-1) was degraded by strain XJ-412-1 after incubation at 30°C for 7 days. The initial degradation products of metsulfuron-methyl (MSM), thifensulfuron-methyl (TSM), and bensulfuron-methyl (BSM) by XJ-412-1 were identified as corresponding deesterified derivatives by liquid chromatography-mass spectrometry, which indicated a primary pathway of the deesterification of these three sulfonylurea herbicides. The carboxyesterase activity of the cell-free extracts was assayed and strongly inhibited by 4-chloromercuribenzoic acid (PCMB), diethyl pyrocarbonate (DEPC), phenylmethylsulfonyl fluoride (PMSF), and malathion.

pH-dependent perforation of macrophage phagosomes by listeriolysin O from Listeria monocytogenes.

The pore-forming toxin listeriolysin O (LLO) is a major virulence factor implicated in escape of Listeria monocytogenes from phagocytic vacuoles. Here we describe the pH-dependence of vacuolar perforation by LLO, using the membrane-impermeant fluorophore 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) to monitor the pH and integrity of vacuoles in mouse bone marrow-derived macrophages. Perforation was observed when acidic vacuoles containing wild-type L. monocytogenes displayed sudden increases in pH and release of HPTS into the cytosol. These changes were not seen with LLO-deficient mutants. Perforation occurred at acidic vacuolar pH (4.9-6.7) and was reduced in frequency or prevented completely when macrophages were treated with the lysosomotropic agents ammonium chloride or bafilomycin A1. We conclude that acidic pH facilitates LLO activity in vivo.

Auxin Signaling-Mediated Apoplastic pH Modification Functions in Petal Conical Cell Shaping.

The flowers of angiosperm species typically contain specialized conical cells. Although substantial progress has been achieved regarding the mechanisms underlying flower development, little is known about how petal cells achieve final conical shape. Here, we use 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) as a fluorescent pH indicator for analyzing the apoplastic pH of conical cells in Arabidopsis and show that normal conical cell expansion requires auxin signaling and apoplastic pH changes. By combining imaging analysis and genetic and pharmacological experiments, we demonstrate that apoplastic acidification and alkalization correlate with an increase and decrease in tip sharpening of conical cells, respectively. Initial expansion of conical cells is accompanied by decreased apoplastic pH, which is associated with increased auxin signaling. Decreased auxin levels, transport, or signaling abolishes cell wall acidification and causes reduced tip sharpening and heights of conical cells. These findings provide an insight into apoplastic pH regulation of conical cell expansion.

Synthesis and Structure-Activity Relationships of Arylsulfonamides as AIMP2-DX2 Inhibitors for the Development of a Novel Anticancer Therapy.

AIMP2-DX2, a splicing variant of AIMP2, is up-regulated in lung cancer, possesses oncogenic activity, and results in tumorigenesis. Specifically inhibiting the interaction between AIMP2-DX2 and HSP70 to suppress AIMP2-DX2-dependent cancers with small molecules is considered a promising avenue for cancer therapeutics. Optimization of hit BC-DXI-04 (IC50 = 40.1 µM) provided new potent sulfonamide based AIMP2-DX2 inhibitors. Among these, BC-DXI-843 showed improved inhibition against AIMP2-DX2 (IC50 = 0.92 µM) with more than 100-fold selectivity over AIMP2 in a luciferase assay. Several binding assays indicated that this compound effectively induces cancer cell apoptosis by specifically interrupting the interaction between DX2 and HSP70, which leads to the degradation of DX2 via Siah1-mediated ubiquitination. More importantly, BC-DXI-843 demonstrated in vivo efficacy in a tumor xenograft mouse model (H460 cells) at a dosage of 50 mg/kg, suggesting it as a promising lead for development of novel therapeutics targeting AIMP2-DX2 in lung cancer.

Identification and characterization of noncalcemic, tissue-selective, nonsecosteroidal vitamin D receptor modulators.

Vitamin D receptor (VDR) ligands are therapeutic agents for the treatment of psoriasis, osteoporosis, and secondary hyperparathyroidism. VDR ligands also show immense potential as therapeutic agents for autoimmune diseases and cancers of skin, prostate, colon, and breast as well as leukemia. However, the major side effect of VDR ligands that limits their expanded use and clinical development is hypercalcemia that develops as a result of the action of these compounds mainly on intestine. In order to discover VDR ligands with less hypercalcemia liability, we sought to identify tissue-selective VDR modulators (VDRMs) that act as agonists in some cell types and lack activity in others. Here, we describe LY2108491 and LY2109866 as nonsecosteroidal VDRMs that function as potent agonists in keratinocytes, osteoblasts, and peripheral blood mononuclear cells but show poor activity in intestinal cells. Finally, these nonsecosteroidal VDRMs were less calcemic in vivo, and LY2108491 exhibited more than 270-fold improved therapeutic index over the naturally occurring VDR ligand 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in an in vivo preclinical surrogate model of psoriasis.

Cytoplasmic pH of Dictyostelium discoideum amebae during early development: identification of two cell subpopulations before the aggregation stage.

Development of the cellular slime mold Dictyostelium discoideum is initiated by the removal of nutrients, and results in formation of a mature fruiting body composed of two cell types, the stalk and spore cells. A considerable body of evidence supports the hypothesis that cytoplasmic pH may be an essential regulator of the choice to differentiate in either the prestalk or prespore pathway. We have devised methods for measurement and analysis of intracellular pH in developing Dictyostelium amebae in order to assess directly the potential role of cytoplasmic pH in regulating the pathway of differentiation. The intracellular pH of single D. discoideum amebae during development and in intact slugs has been measured using the pH-sensitive indicator pyranine in a low light level microspectrofluorometer. We have used the ATP-mediated loading method to introduce pyranine into these cells. Cells loaded by the ATP method appear healthy, have no detectable defects in development, and exhibit a similar population distribution of intracellular pH to those loaded by sonication. The intracellular pH of populations comprised of single amebae was found to undergo a transient acidification during development resulting in a bimodal distribution of intracellular pH. The subpopulations were characterized by fitting two gaussian distributions to the data. The number of cells in the acidic intracellular pH subpopulation reached a maximum 4 h after initiation of development, and had returned to a low level by 7 h of development. In addition, a random sample of single amebae within a slug had a median intracellular pH of 7.2, nearly identical to the median pH (7.19) of similarly treated vegetative cells. No gradient of intracellular pH along the anterior to posterior axis of the slug was detected. Our data demonstrate the existence of two distinct subpopulations of cells before the aggregation stage of development in Dictyostelium, and offers support for the hypothesis that changes in intracellular pH contribute to development in D. discoideum.

Sulfonate desulfurization in Rhodococcus from wheat rhizosphere communities.

Organically bound sulfur makes up about 90% of the total sulfur in soils, with sulfonates often the dominant fraction. Actinobacteria affiliated to the genus Rhodococcus were able to desulfonate arylsulfonates in wheat rhizospheres from the Broadbalk long-term field wheat experiment, which includes plots treated with inorganic fertilizer with and without sulfate, with farmyard manure, and unfertilized plots. Direct isolation of desulfonating rhizobacteria yielded Rhodococcus strains which grew well with a range of sulfonates, and contained the asfAB genes, known to be involved in sulfonate desulfurization by bacteria. Expression of asfA in vitro increased >100-fold during growth of the Rhodococcus isolates with toluenesulfonate as sulfur source, compared with growth with sulfate. By contrast, the closely related Rhodococcus erythropolis and Rhodococcus opacus type strains had no desulfonating activity and did not contain asfA homologues. The overall actinobacterial community structure in wheat rhizospheres was influenced by the sulfur fertilization regime, as shown by specific denaturing gradient gel electrophoresis of PCR amplified 16S rRNA gene fragments, and asfAB clone library analysis identified nine different asfAB genotypes closely affiliated to the Rhodococcus isolates. However, asfAB-based multiplex restriction fragment length polymorphism (RFLP)/terminal-RFLP analysis of wheat rhizosphere communities revealed only slight differences between the fertilization regimes, suggesting that the desulfonating Rhodococcus community does not specifically respond to changes in sulfate supply.

Fatty acid flip-flop in a model membrane is faster than desorption into the aqueous phase.

Fatty acids (FA) are known to diffuse (flip-flop) rapidly across protein-free phospholipid bilayers in their un-ionized form. However, whether flip-flop through the hydrophobic core of the bilayer or desorption from the membrane into the aqueous phase is the rate-limiting step in FA transport through membranes is still debated. The issue has remained unresolved in part by disagreements over whether some methods of adding FA create artifacts that lead to erroneous conclusions and in part by the lack of fluorescence methods to monitor each individual step. Here we study the kinetics of FA transfer from donors to phospholipid vesicles (small and large unilamellar vesicles) by a dual fluorescence approach that utilizes the probes fluorescein phosphatidylethanolamine (FPE) and pyranine. FPE detects the concentration of FA anions in the outer membrane leaflet, allowing a precise measurement of kinetics of FA adsorption or desorption. Our results showed that as soon as FPE detects adsorption of FA into the outer leaflet, pyranine detects its movement to the inner leaflet. We further demonstrated that (i) flip-flop for FA with 14-22 carbons is much faster than the rates of desorption and therefore cannot be the rate-limiting step of FA translocation across membranes; (ii) fluorescence changes detected by probes located on or in acceptor vesicles are dependent upon the method used to deliver the FA (i.e., uncomplexed, or complexed to albumin or phospholipid bilayers); however, (iii) transfer kinetics observed in the presence of different donors is rate-limited by the desorption of FA from the donor into the aqueous phase rather than by flip-flop.

Novel DNA lesions generated by the interaction between therapeutic thiopurines and UVA light.

The therapeutic effect of the thiopurines, 6-thioguanine (6-TG), 6-mercaptopurine, and its prodrug azathioprine, depends on the incorporation of 6-TG into cellular DNA. Unlike normal DNA bases, 6-TG absorbs UVA radiation, and UVA-mediated photochemical damage of DNA 6-TG has potentially harmful side effects. When free 6-TG is UVA irradiated in solution in the presence of molecular oxygen, reactive oxygen species are generated and 6-TG is oxidized to guanine-6-sulfonate (G(SO3)) and guanine-6-thioguanine in reactions involving singlet oxygen. This conversion is prevented by antioxidants, including the dietary vitamin ascorbate. DNA G(SO3) is also the major photoproduct of 6-TG in DNA and it can be selectively introduced into DNA or oligonucleotides in vitro by mild chemical oxidation. Thermal stability measurements indicate that G(SO3) does not form stable base pairs with any of the normal DNA bases in duplex oligonucleotides and is a powerful block for elongation by Klenow DNA polymerase in primer extension experiments. In cultured human cells, DNA damage produced by 6-TG and UVA treatment is associated with replication inhibition and provokes a p53-dependent DNA damage response.

Anionic selectivity sequence of the Cl(-)-H+ symporter in the synaptosomal preparation from rat brain cortex.

The Na(+)/H(+) exchanger has been the only unequivocally demonstrated H(+)-transport mechanism in the synaptosomal preparation. We had previously suggested that a Cl(-)-H(+) symporter (in its acidifying mode) is involved in cytosolic pH regulation in the synaptosomal preparation. Supporting this suggestion, we now show that: (1) when synaptosomes are transferred from PSS to either gluconate or sulfate solutions, the Fura-2 ratio remains stable instead of increasing as it does in 50 mM K solution. This indicates that these anions do not promote a plasma membrane depolarization. (2) Based in the recovery rate from the cytosolic alkalinization, the anionic selectivity of the Cl(-)-H(+) symporter is NO(3)(-) > Br(-) > Cl(-) >> I(-) = isethionate = sulfate = methanesulfonate = gluconate. (3) PCMB 10 muM inhibits the gluconate-dependent alkalinization by 30 +/- 6%. (4) Neither Niflumic acid, 9AC, Bumetanide nor CCCP inhibits the recovery from the cytosolic alkalinization.

Residues of 14C-metsulfuron-methyl in Chinese paddy soils.

BACKGROUND: Metsulfuron-methyl is widely used for controlling many annual grasses and broadleaf weeds in cereal crops. Nonetheless, increasing evidence has demonstrated that even extremely low levels of metsulfuron-methyl residues in soil can be toxic to subsequent crops or non-target organisms. The behavior of herbicides in soils is mostly related to their residual forms. The intent of the present study was to investigate the dynamics of extractable residues (ERs) and non-extractable residues (NERs) of (14)C-metsulfuron-methyl in twelve Chinese paddy soils and their relationships to soil properties. RESULTS: ERs decreased gradually after application, whereas NERs increased rapidly during the initial 28 days, and gradually decreased thereafter. ERs and NERs were respectively 10.1-67.9% and 5.6-28.7% of applied radioactivity in soils at 224 days after application. ERs correlated positively with soil pH and silt fractions, and negatively with microbial biomass carbon (MBC) and clay fractions, but the opposite was observed for NERs. CONCLUSION: Both ERs and NERs may be present in the soil at the time of planting following rice crops, and the risk of phytotoxic effects needs to be considered. Soil pH, MBC and clay/silt fractions were the main factors in affecting the amounts of both ERs and NERs of metsulfuron-methyl in the tested soils.

Phototransformation of herbicide metsulfuron methyl.

Phototransformation of the herbicide metsulfuron methyl was investigated on glass surface under sunlight and ultraviolet (UV) light and compared with dark condition. The half-lives of metsulfuron methyl under UV light and sunlight were found to be 0.5 and 7.8 days respectively. Rate of phototransformation followed first order kinetics with significant correlation coefficient. The major photoproducts were identified as methyl-2-sulfonyl-amino-benzoate, 2-amino-6-methoxy-4-methyltriazine and saccharin (O-sulfobenzoimide). Various metabolites from this study were identified by high performance liquid chromatography (HPLC). Authentic samples required for HPLC comparison were prepared in laboratory and characterized on the basis of nuclear magnetic resonance (NMR) and infra red (IR) spectral data. These metabolites were also identified from metsulfuron methyl treated wheat field soil.