Molecular mechanism of diclofenac sodium on the toxicity of Bacillus thuringiensis
【Abstract】To investigate the molecular toxicity of diclofenac sodium to Bacillus thuringiensis, the iTRAQ quantitative proteomics technique was used to identify and quantify the protein expression of this species. The results showed that diclofenac sodium had significant inhibitory effect on the growth of B. thuringiensis. Seventeen proteins were differentially expressed. Among these proteins, five up-regulated ones were mainly involved in fatty acid biosynthesis as well as DNA and RNA synthesis, while 12 down-regulated proteins were primarily associated with oxidative phosphorylation, pyruvate metabolism, glycolytic pathway, pentose phosphate pathway, and amino acid metabolism. Functional analysis revealed that diclofenac inhibited the growth of B. thuringiensis by affecting cell metabolism, cellular composition, and protein catalysis. In the interaction network of differentially expressed proteins, RpoA, RplM, RplL, Tuf, and InfA were the key nodes in the network interacting closely for cellular regulation. The results indicated that diclofenac sodium could affect multiple metabolic pathways and interfere with different biological processes. These findings revealed the molecular toxicity of diclofenac sodium, and provided important references for the further evaluation of diclofenac sodium on the ecological security and human health.
【Keywords】 diclofenac sodium; Bacillus thuringiensis; differential expressed protein; toxic; molecular mechanism;
 Ji K, Liu X, Lee S, et al. Effects of non-steroidal antiinflammatory drugs on hormones and genes of the hypothalamicpituitary-gonad axis, and reproduction of zebrafish [J]. Journal of Hazardous Materials, 2013, 254: 242–251.
 Acuna V, Ginebreda A, Mor J R, et al. Balancing the health benefits and environmental risks of pharmaceuticals: diclofenac as an example [J]. Environment International, 2015, 85: 327–333.
 Shi R, Zhang F S. Treatment of waste diclofenac sodium medicine by base-catalyzed hydrothermal oxidation method [J]. China Environmental Science, 2017, 37(4): 1386–1393 (in Chinese).
 Yu H, Nie E, Xu J, et al. Degradation of diclofenac by advanced oxidation and reduction processes: kinetic studies, degradation pathways and toxicity assessments [J]. Water Research, 2013, 47 (5): 1909–1918.
 Lepper P. Manual on the methodological framework to derive environmental quality standards for priority substances in accordance with Article 16of the Water Framework Directive (2000/60/EC) [C]//Schmallenberg, Germany: FraunhoferInstitute Molecular Biology and Applied Ecology, 2005.
 Wang Y, Xiong Z H, Zhou J G. Removal of diclofenac on calyx  arene based Amberlite XAD-4 resin from aqueous solutions [J]. China Environmental Science, 2012, 32(1): 81–88 (in Chinese).
 Oaks J L, Gilbert M, Virani M Z, et al. Diclofenac residues as the cause of vulture population decline in Pakistan [J]. Nature, 2004, 427 (6975): 630–633.
 Triebskorn R, Casper H, Heyd A, et al. Toxic effects of the nonsteroidal anti-inflammatory drug diclofenac: Part II. Cytological effects in liver, kidney, gills and intestine of rainbow trout (Oncorhynchus mykiss) [J]. Aquatic Toxicology, 2004, 68 (2): 151–166.
 Cleuvers M. Aquatic ecotoxicity of pharmaceuticals including the assessment of combination effects [J]. Toxicology Letters, 2003, 142 (3): 185–194.
 Cleuvers M. Mixture toxicity of the anti-inflammatory drugs diclofenac, ibuprofen, naproxen, and acetylsalicylic acid [J]. Ecotoxicology and Environmental Safety, 2004, 59 (3): 309–315.
 Ungprasert P, Cheungpasitporn W, Crowson C S, et al. Individual non-steroidal anti-inflammatory drugs and risk of acute kidney injury: a systematic review and meta-analysis of observational studies [J]. European Journal of Internal Medicine, 2015, 26 (4): 285–291.
 Fattori V, Borghi S M, Guazelli C F S, et al. Vinpocetine reduces diclofenac-induced acute kidney injury through inhibition of oxidative stress, apoptosis, cytokine production, and NF-kappa Bactivation in mice [J]. Pharmacological research, 2017, 120: 10–22.
 Syed M, Skonberg C, Hansen S H. Mitochondrial toxicity of diclofenac and its metabolites via inhibition of oxidative phosphorylation (ATP synthesis) in rat liver mitochondria: possible role in drug induced liver injury (DILI) [J]. Toxicology in Vitro, 2016, 31: 93–102.
 Hickey E J, Raje R R, Redd V E, et al. Diclofenac induced in vivo nephrotoxicity may involve oxidative stress-mediated massive genomic DNA fragmentation and apoptotic cell death [J]. Free Radical Biology and Medicine, 2001, 31 (2): 139–152.
 Ng L E, Vincent A S, Halliwell B, et al. Action of diclofenae on kidney mitochondria and cells [J]. Biochemical and Biophysical Research Communications, 2006, 348 (2): 494–500.
 Fatta-Kassinos D, Hapeshi E, Achilleos A, et al. Existence of pharmaceutical compounds in tertiary treated urban wastewater that is utilized for reuse applications [J]. Water Resources Management, 2011, 25 (4): 1183–1193.
 Zorita S, Martensson L, Mathiasson L. Occurrence and removal of pharmaceuticals in a municipal sewage treatment system in the south of Sweden [J]. Science of the Total Environment, 2009, 407 (8): 2760–2770.
 Kambiranda D, Katam R, Basha S M, et al. iTRAQ-based quantitative proteomics of developing and ripening muscadine grape berry [J]. Journal of Proteome Research, 2014, 13 (2): 555–569.
 Zhang L H. Proteomic analysis of zinc effects on Streptococcus pneumoniae [D]. Guangdong: Jinan University, 2010 (in Chinese).
 Huang Y Y, Bai Y, Wang Y, et al. Differentially expressed proteins in Microcystic aeruginosa with Solidago canadensis L. extracts using iTraq labeling technique [J]. China Environmental Science, 2015, 35(6): 1822–1830 (in Chinese).
 Mc Kenney P T, Driks A, Eichenberger P. The Bacillus subtilis endospore:assembly and functions of the multilayered coat [J]. Nature Reviews Microbiology, 2013, 11 (1): 33–44.
 Ozin A J, Henriques A O, Yi H, et al. Morphogenetic proteins SpoVID and SafA form a complex during assembly of the Bacillus subtilis spore coat [J]. Journal of Bacteriology, 2000, 182 (7): 1828–1833.
 Chen F C, Shen L F, Tsai M C, et al. The Isp A protease’s involvement in the regulation of the sporulation process of Bacillus thuringiensis is revealed by proteomic analysis [J]. Biochemical and Biophysical Research Communications, 2003, 312 (3): 708–715.
 Huang J C, Wang X R, Cao Q, et al. Clp P participates in stress tolerance and negatively regulates biofilm formation in Haemophilus parasuis [J]. Veterinary Microbiology, 2016, 182: 141–149.
 Dorel C, Lejeune P, Rodrigue A. The Cpx system of Escherichia coli, a strategic signaling pathway for confronting adverse conditions and for settling biofilm communities [J]. Research in Microbiology, 2006, 157 (4): 306–314.
 Silva A J, Parker W B, Allan P W, et al. Benitez. Role of methylthioadenosine/S-adenosylhomocysteine nucleosidase in Vibrio cholerae cellular communication and biofilm development [J]. Biochemical and Biophysical Research Communications, 2015, 461 (1): 65–69.
 Shimaoka M, Takenaka Y, Kurahashi O, et al. Effect of amplification of desensitized pur F and prs on inosine accumulation in Escherichiacoli [J]. Journal of Bioscience and Bioengineering, 2007, 103 (3): 255–261.
 Shi S B, Shen Z, Chen X, et al. Increased production of riboflavin by metabolic engineering of the purine pathway in Bacillus subtilis [J]. Biochemical Engineering Journal, 2009, 46 (1): 28–33.
 Yin Y L, Ashihara H. Phosphate levels and expression of phosphoribosylpyrophosphate synthetase isozymes in suspension-cultured Arabidopsis thaliana cells [J]. Phytochemistry Letters, 2009, 2 (3): 126–129.
 Chen P. The structural and functional research of human phosphoribosyl pyrophosphate synthase PRS1 and its related proteins [D]. Hefei: University of Science and Technology of China, 2013 (in Chinese).
 Kovacs D, Rakacs M, Agoston B, et al. Janus chaperones: assistance of both RNA-and protein-folding by ribosomal proteins [J]. Febs Letters, 2009, 583 (1): 88–92.
 Kim T Y, Ha C, Huh W K. Differential subcellular localization of ribosomal protein L7paralogs in Saccharomyces cerevisiae [J]. Molecules and Cells, 2009, 27 (5): 539–546.
 Husnain S I, Meng W M, Busby S J W, et al. Escherichia coli can tolerate insertions of up to 16amino acids in the RNA polymerase alpha subunit inter-domain linker [J]. Biochimica Et Biophysica Acta-Gene Structure and Expression, 2004, 1678 (1): 47–56.
 Abushahba M F N, Mohammad H, Seleem M N. Targeting multidrug-resistant staphylococci with an anti-rpo A peptide nucleic acid conjugated to the HIV-1TAT cell penetrating peptide [J]. Molecular Therapy-Nucleic Acids, 2016, 5:e339. https://doi.org/10.1038/mtna.2016.53.
 Cai L, Sutter B M, Li B, et al. Acetyl-Co A induces cell growth and proliferation by promoting the acetylation of histones at growth genes [J]. Molecular Cell, 2011, 42 (4): 426–437.
 Li Y. Optimization of recombinant Bacillus thuringiensis pyruvate dehydrogenase expression and structure prediction [D]. Wuhan: Central China Normal University, 2008 (in Chinese).
 Fleige T, Pfaff N, Gross U, et al. Localisation of gluconeogenesis and tricarboxylic acid (TCA)-cycle enzymes and first functional analysis of the TCA cycle in Toxoplasma gondii [J]. International Journal for Parasitology, 2008, 38 (10): 1121–1132.
 Battaile K P, Molin-Case J, Paschke R, et al. Crystal structure of rat short chain acyl-CoA dehydrogenase complexed with acetoacetyl-CoA-Comparison with other acyl-Co Adehydrogenases [J]. Journal of Biological Chemistry, 2002, 277 (14): 12200–12207.
 Le W P, Abbas A S, Sprecher H, et al. Long-chain acyl-Co Adehydrogenase is a key enzyme in the mitochondrial betaoxidation of unsaturated fatty acids [J]. Biochimica Et Biophysica Acta-Molecular and Cell Biology of Lipids, 2000, 1485 (2/3): 121–128.
 Trchounian A. Escherichia coli proton-translocating F0F1-ATPsynthase and its association with solute secondary transporters and/or enzymes of anaerobic oxidation-reduction under fermentation [J]. Biochemical and Biophysical Research Communications, 2004, 315 (4): 1051–1057.