State Key Laboratory of Pollution Control and Resource Reuse, School of the. Environment, Nanjing University, Nanjing, , P.R.

Transcription

1 Supporting Information For Comparison of cytotoxicity and inhibition of membrane ABC transporters induced by MWCNTs with different length and functional groups Jing Yu, Su Liu, Bing Wu, Zhuoyan Shen, Gary N. Cherr,, Xu-xiang Zhang, Mei Li State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, , P.R. China Bodega Marine Laboratory, University of California, Davis, California, USA Departments of Environmental Toxicology and Nutrition, University of California, Davis, California, USA To whom correspondence may be addressed. (B. Wu) or (M. Li). Telephone: Address: 163 Xian Lin Avenue. Nanjing, China. S1

2 (A) (C) (D) (E) (F) Figure S1 TEM images of MWCNTs used in this study. (A) M-L-COOH; M-S-COOH; (C) M-L-OH; (D) M-S-OH; (E) M-L; (F) M-S. S2

3 Figure S2 FTIR spectra of MWCNTs used in this study. the peak at approximate 1714cm -1 can be attributed to the stretching vibration of carbon-oxygen group. S3

4 (A) (C) Figure S3 Comparison of cytotoxicity induced by MWCNTs with different length on HepG2 cells. Cell viability was determined by CCK-8 assay (n=18). (A) MWCNTs with COOH group. MWCNTs with OH group. (C) Pristine MWCNTs. All differences were identified by one-way ANOVA, followed by Tukey post hoc test. A p-value less than 0.05 (*) was significance. S4

5 (A) (C) Figure S4 Comparison of ROS generation induced by MWCNTs with different length on HepG2 cells (n=18). The ROS level was measured by increase of fluorescence values of DCF. Hoechst was used to measure the number of HepG2 remaining in each well and to normalize the fluorescence values of DCF. Relative DCF fluorescence was calculated and compared to the control group. (A) MWCNTs with COOH group. MWCNTs with OH group. (C) Pristine MWCNTs. All differences were identified by one-way ANOVA, followed by Tukey post hoc test. A p-value less than 0.05 (*) was significance. S5

6 (A) (C) Figure S5 Comparison of mitochondrial membrane potential induced by MWCNTs with different length on HepG2 cells (n=18). The mitochondrial membrane potential was determined by JC1 based on its ratio of red and green fluorescence values. (A) MWCNTs with COOH group. MWCNTs with OH group. (C) Pristine MWCNTs. All differences were identified by one-way ANOVA, followed by Tukey post hoc test. A p-value less than 0.05 (*) was significance. S6

7 (A) Figure S6 Lysosome induced by MWCNTs on HepG2 cells, which was measured by the increase of Lysotracker fluorescence values (n=18). (A) Relative Lysotracker fluorescence values after 24h exposure of M-L, M-L-COOH and M-L-OH; Relative Lysotracker fluorescence values after 24h exposure of M-L, M-L-COOH and M-L-OH. All differences were identified by one-way ANOVA, followed by Tukey post hoc test. A p-value less than 0.05 (*) was significance. S7

8 Relative fluorescence value Control * MK571 Figure S7 Influence of MK571 on CAM accumulation (n=18). The concentration of MK571 is 10µM. The difference was identified by t-test. A p-value less than 0.05 (*) was significance. CK MK571 M-L-COOH M-S-COOH M-L-OH M-S-OH M-L M-S Figure S8 CAM accumulation in HepG2 after a exposure of the six MWCNTs. CAM fluorescence images of HepG2 were captured using an inverted fluorescence microscope. MK571 was used as the positive control. The concentration of MK571 is 10µM. The concentration of M-L, M-L-COOH and M-OH was 0.05µg/mL and M-S, M-S-COOH and M-S-OH was 0.01µg/mL. S8

9 (A) Figure S9 Comparison of influence of MWCNTs with same length on As toxicity (n=18). The generation of ROS was chosen as the toxic endpoint which was measured by cell dye DCF. Two strategies of co-exposure were utilized. The first strategy was to add As at the beginning of exposure; after 12h exposure, MWCNTs were added for another 12h co-exposure. The second strategy was to add MWCNTs at the beginning of exposure; after 12h, the As was added. (A) Comparison among M-L, M-L-COOH and M-L-OH by above two strategies. As+MWCNTs indicates the As was firstly added, and MWCNTs+As indicates the MWCNTs were firstly added. (D) Comparison among M-S, M-S-COOH and M-S-OH by above two strategies. The concentration of M-L, M-L-COOH and M-OH was 0.05µg/mL and M-S, M-S-COOH and M-S-OH was 0.01µg/mL. All differences were identified by one-way ANOVA, followed by Tukey post hoc test. A p-value less than 0.05 (*) was significance. S9

10 Figure S10 Expression of the ABCC transporter MRP2 in HepG2 cells exposed to MWCNTs for 24h. The exposure concentrations for long and short MWCNTs were 0.05 and 0.01µg/mL, respectively. The data was from quantification of Western blotting analysis (n=3). The criteria that fold change >±1.5 and p-value <0.05 was chosen to identify the differently expressed protein. The differences were identified by one-way ANOVA, followed by Tukey post hoc test. A p-value less than 0.05 was significance. S10