Supplemental Information
Molecular Mechanism Comparison between Nano Zinc Oxide (ZnO) Particles and Free
Zinc Ion using a Genome-wide Toxicogenomics Approach
Guanyong Su
1
, Xiaowei Zhang
1,*
, John P. Giesy
1,2,3,4
, Javed Musarrat
4
, Quaiser Saquib
4
,
Abdulaziz A. Alkhedhairy
4
, Hongxia Yu
1,*
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 Zn
2+
/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
Vive Nano Zinc Oxide Powder
ZnCl2 mg/L as Zn
ECx
Estimate Standard Error
EC20 92.1
EC50 321
EC90 2330
7.1
22.7
398
EC20 23.7
EC50 41.0
EC90 97.5
3.4
2.9
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 Pvalue OddsRatio ExpCount Count Size Term
1 GO:0005198 <0.001
2 GO:0003735 <0.001
50.4
50.4
5.67
5.67
12 17 structural molecule activity
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 powder
Concentration of released zinc
50000.00 16666.67 5555.56 1851.85 617.28 205.76 68.59
278.63±20.
99
260.84±2
4.8
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 o
C. The fitted curve was conducted following Langmuir equation, which was simply expressed as: y=0.00316x+20.82, adjusted
R
2
=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 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 Zn 2+ /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.