Supplemental Information

Molecular Mechanism Comparison between Nano Zinc Oxide (ZnO) Particles and Free

Zinc Ion using a Genome-wide Toxicogenomics Approach

Guanyong Su1, Xiaowei Zhang1,*, John P. Giesy1,2,3,4, Javed Musarrat4, Quaiser Saquib4,

Abdulaziz A. Alkhedhairy4, Hongxia Yu1,*

1 State Key Laboratory of Pollution Control and Resource Reuse & School of the

Environment, Nanjing University, Nanjing, China

2 Department of Biomedical Veterinary Sciences and Toxicology Centre, University of

Saskatchewan, Saskatoon, SK S7N 5B3, Canada

3 Department of Biology & Chemistry, City University of Hong Kong, 83 Tat Chee

Avenue, Kowloom, Hong Kong SAR, China

4 Zoology Department, College of Science, King Saud University, P.O. Box 2455, Riyadh

11451, Saudi Arabia

Authors for correspondence:

School of the Environment

Nanjing University

Nanjing, 210089, China

Tel: 86-25-83593649

Fax: 86-25-83707304

E-mail:

zhangxw@nju.edu.cn (Xiaowei Zhang) & yuhx@nju.edu.cn (Hongxia Yu)

Supplementary Data description

Table S1 Acute toxicity endpoint of nano-ZnO and zinc ion

Table S2 Gene Set Enrichment Analysis on 387 selected genes to nano-ZnO against the genome-wide live cell array library. The association of gene ontology was performed by R 3.0.2 version using “GOstats” package. The universe and selected genes were defined according 1820 genes in the live cell array library and 387 selected differently expressed genes, respectively. The analysis was conducted basing on the cell components (CC) GO ontology.

Table S3, Concentration of released Zinc as measured by a serial dilution of nano-ZnO solution (unit: mg/L)

Figure S1 Transmission electron micrographs of nano-ZnO (provided by Vive nano team)

Figure S2 Size distribution of nano-ZnO

Figure S3 Relationships between the released zinc ion and nano-ZnO

Figure S4 Concentrations of determined released zinc in fresh LB medium and 100 mg/L nano-ZnO spiked LB medium. (Here, 0 and 6 h mean incubation time points when the free zinc ion concentrations were determined. N=3 replicates)

Figure S5 Real-time, quantitative determination of gene expression as measures of differentially expressed promoter activities in E. coli following exposures to 0.06, 0.6 and 6 mg Zn2+/L. Classification and visualization of the gene expression were derived by use of ToxClust. The dissimilarity between genes was calculated by the Manhattan distance between the gene expressions at all the concentration vs. time combinations. The fold change of gene expression is indicated by color gradient, and the time course of expression changes is indicated from left to right.

Table S1, Acute cytotoxicity of Zn in Vive Nano Zinc Oxide Powder & Zinc chloride

　 ECxmg/L as Zn

Estimate Standard Error

Vive Nano Zinc Oxide Powder

EC20

92.1 7.1

EC50

321 22.7

EC90

2330 398

ZnCl2

EC20

23.7 3.4

EC50

41.0 2.9

EC90

97.5 14.4

Table S2 Gene Set Enrichment Analysis on 387 selected genes to nano-ZnO against the genome-wide live cell array library. The association of gene ontology was performed by R 3.0.2 version using “GOstats” package. The universe and selected genes were defined according 1820 genes in the live cell array library and 387 selected differently expressed genes, respectively. The analysis was conducted basing on the cell components (CC) GO ontology.

　 GOMFID PvalueOddsRati

oExpCount

Count

Size

Term

1GO:000519

8<0.001 50.4 5.67 12 17 structural molecule activity

2GO:000373

5<0.001 50.4 5.67 12 17 structural constituent of ribosome

Table S3, Concentration of released Zinc as measured by a serial dilution of nano-ZnO solution (unit: mg/L)Concentration of VNZO

powder50000.00 16666.67 5555.56 1851.85 617.28 205.76 68.59

Concentration of released zinc

278.63±20.99

260.84±24.8

95.05±5.82

52.16±0.17

29.54±0.91

12.84±1.36

8.26±3.44

Figure S1, Transmission electron micrographs of nano-ZnO (provided by producer)

Figure S2 Size distribution of nano-ZnO

A: Concentration: 10 mg/L

B: Concentration: 100 mg/L

C: Concentration: 1000 mg/L

D: Concentration: 10000 mg/L

Figure S3, Concentrations of released zinc in a serial of nano-ZnO (Before the Zn content determination, the nano-ZnO (50000, 16666.67, 5555.56, 1851.85, 617.28, 205.76 and 68.59 mg/L) were incubated for 6 h at a temperature of 37 oC. The fitted curve was conducted following Langmuir equation, which was simply expressed as: y=0.00316x+20.82, adjusted R2 =0.9557.)

Figure S4 Concentrations of determined released zinc in fresh LB medium and 100 mg/L nano-ZnO spiked LB medium. (Here, 0 and 6 h mean incubation time points when the free zinc ion concentrations were determined. N=3 replicates)

Figure S5Real-time, quantitative determination of gene expression as measures of differentially expressed promoter activities in E. coli following exposures to 0.06, 0.6 and 6 mg Zn2+/L. Classification and visualization of the gene expression were derived by use of ToxClust. The dissimilarity between genes was calculated by the Manhattan distance between the gene expressions at all the concentration vs. time combinations. The fold change of gene expression is indicated by color gradient, and the time course of expression changes is indicated from left to right.