Analysis of GC-MS Rice Metabolomics Data

Integratedion pathway-fixed and network-fixed segregation of GC-MS Rice metabolomics postulates below Diazinon Emphasis with Metaboanalyst and Metabolic Networks Reveals Follower Reactions

  • Vahideh Mahdavi
  • Zhaleh Hosseini
  • Sayed-Amir Marashi
  • Alireza Ghassempour


Diazinon insecticide is amplely applied throghextinguished rice (Oryza sativa L.) arenas in Iran. However, concerns are now life exorbitant environing its possible impertinent contacts on rice arenas. In this consider, a era-course metabolic qualify in rice fixs was questiond behind diazinon tenor using fog chromatography−magnitude spectrometry (GC−MS) and subsequentely, three incongruous manners were truthd in classify to integadmonish qualifys in metabolite levels to changes in biological manners, namely metaboanalyst, metabonetwork, and segregation of follower reactions with metabolic networks.statistical diplomacy of metaboanalyst, metabolic networks and follower reactions was executed in classify to perceive the emphasis-associated property. According to the upshots, a ample class of metabolites were dynamically numerous as a upshot of the fix retort to diazinon such as biosynthesis and metabolism of sugars, amino fines, radical fines and phenylpropanoids, complete correlating with the pitftotal era. Fix retort was concerned in multiple metabolic manners, such as tricarboxylic fine cycle (TCA cycle), shikimate manner, and biosynthesis of amino fines, most of which were correlated with the pitftotal era.

Key Words: Diazinon, Rice, Metabolomics, Metaboanalyst, Metabolic Networks, Follower Reactions

  1. Introduction

Rice (Oryza sativa L.) is confirmed complete aggravate the earth and is rationalistic in damp and abstemious environments, which makes its evolution capable to fungi, insects and mites. More than 70 insect quality enjoy been recorded as rice scourges, so that this would be considered as individual of the senior constraints on harvest yields causing important decrease in fix evolution. To discourse this gist, unanalogous barks of insecticides, fungicides and herbicides are utilized in classify to secure harvests opposite scourge detriment. The application of scourgeicides in rice arenas has befit a common advent towards guideing scourge detriments during give periods of rice husbandry in Asia [1]. Diazinon (O, O-diethyl O-(2-isopropyl-6-methylpyrimidin-4-yl) thiophosphate, Figure 1) is an organophosphorus scourgeicide (OPP) commercially introduced in 1952 [2]. Thanks to its inhibitory property on acetyl cholinesterase enzyme in a prodigious seniority of insects, Diazinon is universally utilized in urban sectors restraint fix secureion opposite a difference of sucking and leaf-eating insects [3]. Nevertheless, unanalogous newss enjoy demonstrated that diazinon is immunotoxic [4] cytotoxic [5] and genotoxic [6], future it exhibits toxic properties and possible catruth to civilized heartiness. As individual of the most common harvests earthwide, it seems leading to explore the govern of diazinon on metabolite profiling of rice. To this purpose, metabolomics is individual of the most strong dupes restraint providing an aggravateview of metabolite qualifys below diversified abiotic emphasises, namely scourgeicide emphasis[7].

So remote, No news concerning the govern of diazinon on metabolite profiling of rice has been recorded. Therefore, the give consider was belowenthralled to question the commodities of diazinon on rice metabolite profiling below sub-tropical climatic stipulations.

Recently, Even though oral postulates segregation manners affect pre-eminent element segregation, clustering segregation and chemometrics enjoy shown to be efficient restraint segregation of this bark of postulates (Raamsdonk et al, 2001; Completeen et al, 2003), there are some limitations with these manners restraint uncovering the belowlying biological principles (Weckwerth et al, 2004). Furthermore, there are quiet merely reconnoitret examples of studies on the truth of metabolome postulates to belowstand regulatory principles in metabolism. The postulates processing challenges in metabolomics are perfectly uncommon and repeatedly claim specialized (or high-priced) postulates segregation software and a elaboadmonish enlightenment of cheminformatics, bioinformatics and statistics.

Fundamentally, MetaboAnalyst is a web-fixed metabolomic postulates processing dupe that accepts a difference of input postulates (NMR peak lists, binned spectra, MS peak lists, compound/tension postulates) in a ample difference of restraintmats. It also offers a sum of options restraint metabolomic postulates processing, postulates normalization, multivariate statistical segregation, graphing, metabolite identification and manner mapping.

MetaboNetworks is a dupe to invent manner sub-networks in Matlab using main reaction pairs as defined by the Kyoto Encyclopaedia of Genes and Genomes(KEGG) and can be investigate the shortest route betwixt a established of metabolites and plots the connectivity betwixt metabolites as integrates in a netachievement graph.

Follower reaction segregation is an try to argue the incongruousial reaction opinion fixed on metabolite measurements, and future provides a foundation restraint belowstanding the belowlying cellular processes responding to the perturbations.

In this consider, restraint the primeval, we enjoy explored metaboanalyst, metabolic networks and follower reactions as a possible multivariate manner restraint GC/MS segregation of metabolomics postulates of rice (Oryza sativa L.) fixs below diazinon emphasis. More than 31 metabolites of sugars, amino fines and radical fines were quantitavely steadfast and era-course metabolic retort of the fix during incongruous post-tenor days were questiond and measured in berestraint achievement.

2. Materials and Manners

2.1. Materials

Seeds of Shiroodi difference (Oryza sativa ssp. indica) were obtained from Rice Research Institute of Iran and were subsequentely rationalistic in the similar disclosed arena to coercionsake the govern of growing residuum. Diazinon was industrious to coercionefend the fix from insect detriment, conjuncture its applied tension was fixed on the recommended clear dosage from Fix Secureion Organization (PPO). Fixs were subjected to 10% granular restraintmulation of diazinon(15 kghectare-1) during address and blossoming era. Untreated fixs were fixed below the similar experimental stipulations. Rice leaves were enthralled from guide and treated fixs at 24, 48, 96 and 120 h behind tenor. Seven replicates at each era top were self-possessed and presently chilled in glideing nitrogen. Frozen leaves were manually account in a mortar, using glideing nitrogen in oredr to adhere-to specimens at cryogenic sky and eventuallty complete specimens were stored at −80 °C until metabolite segregation[8].

2.2. Chemicals

HPLC-grade methanol was purchased from Merck concourse (Darmstadt, Germany). Ultrapure impart was free by a Milli-Q plan (Millipore, MA, USA). N-Methy-N-(trimethylsilyl) trifluoroacetamide (MSTFA), methoxyamine hydrochloride, sorbitol, trimethylchlorosilan (TMCS) and pyridine were obtained from Sigma-Aldrich Concourse (Steinheim, Germany).

2.3. Specimen Preparation

Extraction process and derivatization of the metabolits were carried extinguished using a mitigated manner picturesquely by Roessner et al. [9]. Approximately 50 mg of powdered rice leaves were homogenized in 1400 μL of 100% methanol (4 °C), and 50 μL of inner scale (2 mg sorbitolml-1 impart) was acquired. The conformation was then vortexed restraint 15 min at 70 °C. The extol was vigorously partial with 1400 μL impart (4 °C) and astern centrifuged restraint 10 min at 2200 g. The polar metabolite fragment containing aliquots of the methanol/impart (1000 μL) was infections to an eppendorf tube and dried in vacum restraint 6-16 h. A co-operation of oxidation and silylation reactions was conducted as derivatization process. Primeval, dried remainder was dissolved and derivatized via adding 80 μL of methoxyamine (20 mgml-1 in pyridine) answer and incubated in 30 °C impart bath restraint 90 min. Then, 80 μL of MSTFA and 0.8 μL TMCS (1% MSTFA) were acquired restraint trimethylsilylation (37 °C restraint 30 min).

2.4. GC−MS Segregation

A Varian ion snare MS 4000 and electron contact ionization competition was coupled with a Varian cp-3800 fog chromatograph. MS coercionward outoutline and ion snare skys were established at 270 and 200 â-¦C, respectively. Individual μL of derivatized specimen was injected into fog chromatography (GC) and metabolites were divided on a 30 m × 0.25 mm × 0.25 μm DB-5 MS post. Injection sky was established at 250 °C and the ion cause sky was adjusted to 200 °C. Helium was truthd as consigner fog at a glide admonish of 1 mLmin-1. Segregation was executed below the coercionthcoming sky program: 5 min of isothermal heating at 70 °C, followed by a 5 °Cmin-1 patch restraint oven sky to 310 °C and a decisive 10-min heating at 310 °C. Ion snare was operated in ample reconnoitre statute at magnitude class of 50650 m/z. Both chromatograms and magnitude spectra were evaluated using the GC–MS Postrun Segregation (Varian).

2.5. Data Processing

Metabolomic postulates processing and statistical segregation was executed restraint the GC-MS chromatograms by coercionthcoming the achievementglide of MetaboAnalyst 2.0, a web-fixed all dupe retainers restraint metabolomic postulates segregation (


[1] G. Matthews, R. Bateman, and P. Miller, Scourgeicide application manners, John Wiley & Sons, 2014.

[2] P. Li, E. Swanson, and F. Gobas, Diazinon and its suspension products in urban impart courses in British Columbia, Canada. Bulletin of environmental defilement and toxicology 69 (2002) 59-65.

[3] Y. Zhang, W. Zhang, X. Liao, J. Zhang, Y. Hou, Z. Xiao, F. Chen, and X. Hu, Suspension of diazinon in apple juice by ultrasonic tenor. Ultrasonics sonochemistry 17 (2010) 662-668.

[4] E.Z. Neishabouri, Z. Hassan, E. Azizi, and S. Ostad, Evaluation of immunotoxicity requisite by diazinon in C57bl/6 mice. Toxicology 196 (2004) 173-179.

[5] Y. Zhang, Y. Hou, F. Chen, Z. Xiao, J. Zhang, and X. Hu, The suspension of chlorpyrifos and diazinon in aqueous answer by ultrasonic irradiation: Commodities of parameters and suspension manner. Chemosphere 82 (2011) 1109-1115.

[6] Åž. Çakir, and R. Sarikaya, Genotoxicity testing of some organophosphate insecticides in the< i> Drosophila

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