Ganesan Raja, Siwon Kim, Dahye Yoon, Heonho Lee and Suhkmann Kim* Metabolomic study of TiO2 nanoparticles exposed to zebrafish (Danio rerio) using 1H-NMR spectroscopy method Structural Biochemistry Lab. in PNU Ganesan Raja, Siwon Kim, Dahye Yoon, Heonho Lee and Suhkmann Kim* Department of Chemistry, Center for Proteome Biophysics, Pusan National University, Busan, 609-735, Korea ABSTRACT Materials & methods Sample preparation 1H NMR Experiments Multivariate analysis & Assignment Zebrafish (Danio rerio) Tap water – control, TiO2 (3 groups)- 24, 48, 72hrs Temp = 26 ± 1°C, Light : Dark = 8:16 Flash-frozen in Liquid N2 and lyophilized Extraction with Methanol : water = 1.6 : 0.6 Chloroform 0.8 ml Chloroform : water = 1: 1 Aqueous extracts – D2O 700µl (TSP-d4 2mM) Depurate for 1 hr Homogenize Lyophilize NMR chemical shift measurement The oral toxicological effects of TiO2 nanoparticles (dosed at 0mg/L, 0.1mg/L, 5.0 mg/L respectively) were investigated using target approaches and metabolomic analysis in zebrafish. We are conducted a comprehensive TiO2 toxicity assessment at 24hrs, 48hrs and 72hrs toxicity test and also accumulation analysis. The concentrated TiO2 nanoparticles dispersed solutions were prepared by sonication without any solvents. It was demonstrated that chronic exposure of zebrafish to 0.1mg/L TiO2 can significantly impair zebrafish reproduction [1]. Chenomx NMR Suite 7.0 and SIMCA P+ were used for compound targeting and statistical analysis respectively. PLS–DA, OPLS-DA score plots are explained separation between control and TiO2 treated groups. Faria, M et al. was fully explained about TiO2 nanoparticles oxidative stress, ROS production, acute toxicity and accumulation with zebrafish embryos [2]. Oxidative stress is a common pathway of toxicity of many pollutants that may affect organisms through several mechanisms [2]. Ecotoxicological studies with TiO2 NPs are much more limited with few reports on invertebrates, but almost no information on the toxic effects TiO2 NPs to fish [3]. The metabolic signature of samples in 24hrs TiO2 nanoparticles treated zebrafish showed decrease metabolites in the levels of ADP, AMP, ATP, acetate, alanine, arginine, asparagine, aspartate, betaine,..etc; moreover, increases in the levels of glutamate, Inosine, lactate, lysine, oxalcetate, threonine,..etc was observed. At the same time, 48 hrs, 72hrs TiO2 induced metabolites changes were significantly compared with timely repots. These results confirm that in zebrafish samples was significantly influenced by TiO2 exposure. Based on the NMR chemical shift analysis, the result of TiO2 induced metabolites was explained through net work diagram such as hypergraphs and bipartite graphs. Results & Discussion Multivariate analyses Percentages level metabolites (a) (b) Fig 1. OPLS-DA Score Plots to Compare Metabolites Between Control and TiO2 treated groups. OPLS-DA score plot (t1 vs. t2) obtained from the NMR spectra of fish samples using SIMCA-P+ 12.0. Fish samples were classified into three groups. (a) : ●: 48h-control , ●: 48h-0.1mg, : 48h-5mg. (b): ●: 48h-control, : 48h-5mg. Fig 3. 24 Hrs TiO2 differential expression levels (mean) of metabolites in different groups. (a) (b) TiO2 molecular metabolites Fig 2. OPLS-DA Score Plots to Compare Metabolites Between Control and TiO2 treated groups. OPLS-DA score plot (t1 vs. t2) obtained from the NMR spectra of fish samples using SIMCA-P+ 12.0. Fish samples were classified into three groups. (a): ●: 72h-control , ●: 72h-0.1mg, : 72h-5mg. (b): ●: 72h-control , :10mg Fig 4. Summary of biochemical pathways of glucose metabolism affected by TiO2 in muscle and liver at zebrafish. conclusions REFERENCE We are provides most detailed synopsis of NMR metabolomics and the physiological effects of TiO2 nanoparticles in zebrafish. We are studied and resulted the TiO2 nanoparticles toxicity at three different timing intervals. The chenomx nmr suit 7.1 was used for metabolites baseline correction and target profiling. The SIMCA P+ multivariate statistical analysis was explained the difference between control and TiO2 nanoparticles induced metabolites. We are monitored that three different timing metabolites, which has caused significantly more toxicity. We are calculated relative concentration and percentage of each metabolites at all concentration. In 24hrs TiO2 induced metabolites, increased metabolites are glutamate, Inosine, lactate, lysin, oxalacetate, threonine…etc and similarly ADP, AMP, ATP, acetate, alanine, arginine, asparagine, aspartate, betaine are decreased. From the results, cellular metabolite metabolisms are altered by TiO2 nanoparticles. Our metabolomics research group is focusing the cancer cells and zebrafish at various metals, nanoparticles and natural herbal product metabolites. [1] Wang, J et al., Chemosphere 2011, 83(4), 461-467. [2] Faria, M et al., Sci Total Environ. 2014, 470-471, 379-389. [3] Federici, G et al., Aquat Toxicol. 2007, 84(4), 415-430. Structural Biochemistry Lab. in PNU