Comparison of Cytotoxicity and Inhibition of Membrane ABC

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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, and Mei Li Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 04 Mar 2016 Downloaded from http://pubs.acs.org on March 4, 2016

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Environmental Science & Technology

1

Comparison of cytotoxicity and inhibition of membrane

2

ABC transporters induced by MWCNTs with different

3

length and functional groups

4

Jing Yu†, Su Liu†, Bing Wu†, Zhuoyan Shen†, Gary N. Cherr‡,§, Xu-Xiang Zhang†,

5

Mei Li†

6

†

7

Environment, Nanjing University, Nanjing, 210023, P.R. China

8

‡

Bodega Marine Laboratory, University of California, Davis, California, USA

9

§

Departments of Environmental Toxicology and Nutrition, University of California,

State Key Laboratory of Pollution Control and Resource Reuse, School of the

10

Davis, California, USA

11

12

[email protected] (M. Li). Telephone: 0086-25-89680720. Address: 163 Xian Lin

13

Avenue. Nanjing, China.

To whom correspondence may be addressed. E-mail: [email protected] (B. Wu) or

14

1

ACS Paragon Plus Environment


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ABSTRACT

16

Experimental studies indicate that multi-walled carbon nanotubes (MWCNTs) have

17

the potential to induce cytotoxicity. However, the reports are often inconsistent and

18

even contradictory. Additionally, adverse effects of MWCNTs at low concentration

19

are not well understood. In this study, we systemically compared adverse effects of

20

six

21

carboxyl-MWCNTs of two different lengths (0.5-2µm and 10-30µm) on human

22

hepatoma cell line HepG2. Results showed that MWCNTs induced cytotoxicity by

23

increasing reactive oxygen species (ROS) generation and damaging cell function.

24

Pristine short MWCNTs induced higher cytotoxicity than pristine long MWCNTs.

25

Functionalization increased cytotoxicity of long MWCNTs, but reduced cytotoxicity

26

of short MWCNTs. Further, our results indicated that the six MWCNTs, at non-toxic

27

concentration, might not be environmentally safe as they inhibited ABC transporters’

28

efflux capabilities. This inhibition was observed even at very low concentrations,

29

which were 40-1000 times lower than their effective concentrations on cytotoxicity.

30

The inhibition of ABC transporters significantly increased cytotoxicity of arsenic, a

31

known substrate of ABC transporters, indicating a chemosensitizing effect of

32

MWCNTs. Plasma membrane damage was likely the mechanism by which the six

33

MWCNTs inhibited ABC transporter activity. This study provides insight to risk

34

assessments of low levels of MWCNTs in the environment.

35

Keywords: Multi-walled carbon nanotubes, Cytotoxicity, ABC transporter, Arsenic

MWCNTs

including

pristine

MWCNTs,

hydroxyl-MWCNTs

2


and

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36

INTRODUCTION

37

Single or multiple sheets of graphite can be rolled to form seamless cylinders

38

called carbon nanotubes (CNTs). A single sheet forms a single-walled CNT

39

(SWCNT), while multiple sheets form a multi-walled CNT (MWCNT)1. In recent

40

years, CNTs have been synthesized and mass-produced because of their unique

41

optical, electrical, and mechanical properties leading to a broad range of areas from

42

consumer goods to medical applications2. Compared with SWCNTs, MWCNTs have

43

great advantages on practical applications considering their more simple preparation

44

process, lower price and larger production scale. Along with the wide application of

45

MWCNTs, their impacts in the environment have received more and more attention.

46

Toxicities of MWCNTs have been widely analyzed by in vitro and in vivo

47

methods. Most of the currently available data suggest that MWCNTs are cytotoxic

48

and genotoxic3, 4. Oxidative stress and membrane damage are considered as the

49

widely recognized mechanism for MWCNTs-induced toxicity. Recent studies found

50

that the biological reactivity and toxicity of MWCNTs depend on numerous

51

physicochemical characteristics such as length, diameter, surface area, functional

52

groups, and presence and nature of catalyst residues. Among those characteristics,

53

length and functional groups have been proved to play critical roles in the biological

54

reactivity of MWCNTs5, 6. The long MWCNTs (3-14µm) could induce higher

55

cytotoxicity in RAW264.7 cell line than short MWCNTs (1.5µm)7. However, other

56

studies found that short MWCNTs (0.6µm) caused higher toxicity in epithelial cell

57

lines8. Additionally, functional groups can increase the solubility of MWCNTs, which

58

change the toxicity of MWCNTs. However, like the influence of length, results from

59

different studies on the influence of hydroxyl- and carboxyl- groups on MWCNTs

60

toxicity are contradictory9, 10. Most of available studies just analyzed the influence of 3



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length or functional groups on different cell lines or animals. Because of the

62

differences in characteristics of MWCNTs and exposure conditions, it is difficult to

63

directly compare the results from different publications. As such, it is critical to

64

systemically analyze the toxic effects of MWCNTs with different length and

65

functional groups under the same experimental conditions. Recent studies found that the lowest effective concentration of MWCNTs range

66

1, 9, 11

67

from 5 to 20 µg/mL in different cell lines

68

doses of MWCNTs may be environmentally safe. However, our previous studies

69

found that the low doses of zinc oxide nanoparticle and graphene at not-toxic low

70

concentration could inhibit the activity of plasma membrane ATP-binding cassette

71

(ABC) transporters12, 13. In the cell membrane bilayer, ABC transporters play crucial

72

roles in multidrug resistance, which can also pump xenobiotics out cells using an

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ATP-dependent mechanism, thus reducing potential toxic effects14, 15. However, the

74

inhibition of ABC transporter activity induced by zinc oxide nanoparticle and

75

graphene could reduce the efflux of other toxic compounds to make the cell more

76

sensitive to these toxic chemicals, resulting in chemosensitization12, 13. The zinc oxide

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and

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graphene/graphene oxide (two-dimensional nanomaterial) inhibit ABC transporter

79

activity via different mechanisms, where the metal oxides inhibit via competitive

80

substrate while two dimensional hydrophobic materials damage the cell membrane.

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However, for MWCNTs (one-dimensional nanomaterial), no data on their

82

chemosensitizing effects or efflux pump inhibition mechanisms are available. Such

83

information is very important in establishing the environmental risk of MWCNTs and

84

a thorough understanding of the influence of length and functional groups in the

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toxicity of MWCNTs.

copper

oxide

nanoparticles

, and it has been concluded that low

(zero-dimensional

4


nanomaterial)

and

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86

In this study, we choose six MWCNTs including pristine MWCNTs,

87

carboxyl-MWCNTs and hydroxyl-MWCNTs of two lengths (long: 10-30µm; short:

88

0.5-2µm) to compare their toxic effects in human hepatoma cell line HepG2 under

89

high and low exposure levels. Cell viability, oxidative stress, mitochondrial

90

membrane potential, activity of membrane ABC transporters, and chemosensitizing

91

effects of the six MWCNTs were systematically analyzed. Results from this study

92

provides basic information for the risk assessment of MWCNTs in the environment.

MATERIALS AND METHODS

93

94

MWCNT preparation and characterization

95

Six MWCNTs with different length and functionalized groups, including pristine

96

long MWCNT (M-L), pristine short MWCNT (M-S), long hydroxyl-MWCNT

97

(M-L-OH),

98

(M-L-COOH) and short carboxyl-MWCNT (M-S-COOH) were purchased from

99

Nanjing XFNANO Materials Tech Co., Ltd. (China). Basic characteristics of the six

100

MWCNTs are shown in Table 1, which are provided by the manufacture. We further

101

verify their diameter in culture media by transmission electron microscopy (TEM)

102

with a JEM-200CX electron microscope. The functional groups in MWCNTs were

103

analyzed by FTIR spectra, which were conducted with a Thermo Nicolet NEXUS870.

104

Cell culture and exposure of MWCNTs

short

hydroxyl-MWCNT

(M-S-OH),

long

carboxyl-MWCNT

105

The HepG2 cell was purchased from KeyGEN Biotech (China). Cells were

106

maintained in Dulbecco’s modified Eagles medium (DMEM) containing 10% fetal

107

bovine serum (FBS) at 37°C in a humidified atmosphere of 5% CO2. Stock solutions

108

of MWCNTs at approximately 1000 mg/mL were prepared in deionized water by

109

sonication for 30min to ensure proper dispersion. The stock solutions were diluted 5



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with DMEM to achieve final concentrations before characterization and exposure.

111

Before treatment with MWCNTs, the cells were rinsed with PBS, trypsinized, and

112

then transferred on to 96-well plates at a density of 1.0×104 cells/well. After 24h of

113

growth, the cells were exposed to six MWCNTs for 24h, which were used for further

114

analyses.

115

Cell viability

116

Cell viability of HepG2 was determined by cell counting kit-8 (CCK-8, Dojindo

117

Molecular Technologies,

Inc.

Japan).

After 24h

exposure

with

different

118

concentrations of MWCNTs. 10µL of CCK-8 solution was added into each well of the

119

96-well plate, and incubated for 2h at 37oC with 5% CO2. Then, the cells were

120

measured at 450nm with a microplate reader (Synergy H1, BioTek). Cell viability was

121

calculated from the relative absorbance.

122

Intracellular reactive oxygen species (ROS)

123

Intracellular ROS level was measured by cell dye 2,7-dichlorofluorescin

124

diacetate (DCF, Molecular Probes). In order to reduce the errors from loss of cell

125

number during MWCNTs exposure, Hoechst 33342 (YeaSen Bio-technology), a

126

fluorescent dye used to stain DNA, was used to measure the number of HepG2 cells

127

remaining in each well and to normalize the DCF fluorescence value16, 17. After

128

MWCNTs exposure, DCF (final concentration: 10µM) was added into each well of

129

96-well plate. After 25min incubation, the cells were washed by PBS. Then, Hoechst

130

33342 (final concentration: 5µg/mL) was added and incubated for 15min. The

131

fluorescence values of DCF and Hoechst 33342 were measured using a microplate

132

reader (Synergy H1, BioTek). The excitation and emission wavelengths for DCF are

133

485nm and 530nm, respectively, while 350nm and 460nm was used for Hoechst

134

33342. 6


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135 136

Mitochondrial membrane potential Mitochondrial membrane potential, which is an early indicator of cell apoptosis, 18

137

was measured by a JC1 detection kit (KeyGEN, Nanjing)

. After 24h exposure of

138

MWCNTs, JC1 solution (final concentration: 20nM) was added into each well of a

139

96-well plate, and the plate was incubated for 25min. The fluorescence values of JC1

140

were measured by a microplate reader (Synergy H1, BioTek). There are two

141

fluorescence values for JC1. The excitation and emission wavelengths for red

142

fluorescence were 485nm and 530nm, while those for green fluorescence were 530nm

143

and 590nm, respectively. The ratio of red to green fluorescence values was used to

144

indicate the mitochondrial membrane potential.

145

Lysosome

146

Lysosomes in HepG2 cell were determined by Lysotracker® Deep Red

147

(Molecular Probes), which indirectly indicate cell uptake of MWCNTs19. Like the

148

DCF assay, Hoechst 33342 was used to normalize the fluorescence value of

149

Lysotracker. After 24h exposure of MWCNTs, Lysotracker solution (final

150

concentration: 50nM) was added into each well and the cells were incubated for

151

30min. Then Hoechst 33342 was added and incubated as described above. Finally,

152

cells were analyzed on a microplate reader (Synergy H1, BioTek). The excitation and

153

emission wavelengths for Lysosotracker were 647nm and 668nm, respectively.

154

Membrane transporter activity

155

Calcein-AM (CAM, Dojingdo Molecular Technologies, Inc. Japan) was used to

156

indicate ABC transporter activity through a dye accumulation assay20, 21. MK571, a

157

known inhibitor of multidrug resistance associated proteins (MRPs/ABCCs), one

158

subfamily of ABC transporters, was used as the positive control22. After 24h exposure

159

of MWCNTs, CAM with a final concentration of 0.25µM was added to each well of a 7



160

96-well plate. After 2h incubation, the cells were washed with Hanks buffer. Hoechst

161

33342 was also used to normalize the CAM fluorescence values to cell number. The

162

fluorescence values were measured on a microplate reader (Synergy H1, Bioteck).

163

The excitation and emission wavelengths of CAM were 485nm and 530nm,

164

respectively. CAM fluorescence images of HepG2 after MWCNTs exposure were

165

captured using an inverted fluorescence microscope (Nikon Eclipse Ti-U).

166

Co-exposure of MWCNTs and arsenic

167

We investigated whether the inhibition of ABC transporter activity could result in

168

chemosensitization and thus influence toxicity of other pollutants. Arsenic (As), a

169

known substrate of ABCC transporters, was chosen as the target pollutant. Arsenic

170

oxide was obtained from NSI Solution Inc. Two strategies of co-exposure of

171

MWCNTs and As were utilized. The first strategy was to add As at the beginning of

172

exposure; after 12h exposure, MWCNTs were added for another 12h co-exposure.

173

The second strategy was to add MWCNTs at the beginning of exposure; after 12h, the

174

As was added. MK571 was used as the positive control of inhibition of transporter

175

activity, and was added as described above two strategies. Intracellular ROS level was

176

chosen as the toxic endpoint.

177

Membrane fluidity

178

Membrane

fluidity

was

determined

by

4′-(trimethylammonio)-

179

diphenylhexatriene (TMA-DPH) (AAT Bioquest Inc., USA). After 24h exposure of

180

MWCNTs, the TMA-DPH (final concentration: 1.5µM) was added into each well and

181

the cells were incubated for 20min. Polarization ratio of TMA-DPH was measured in

182

a microplate reader (Synergy H1, Bioteck). Membrane fluidity was negative

183

correlated with the value of fluorescence polarization. A background control without

184

the TMA-DPH probe was measured under the same conditions as the samples. 8


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Western blotting

186

Expression levels of MRP2 protein, an important ABCC transporter were

187

determined by Western blotting. After MWCNTs exposure, RIPA Lysis buffer

188

(SunShine Bio, China) was used to extract proteins of HepG2 cell. 100µg of extracted

189

proteins were separated on 10% SDS-PAGE and transferred onto a PVDF membrane.

190

The membrane was blocked with 5% skim milk in TBS buffer with 0.1% Tween 20

191

(pH7.6) for 2h and incubated with primary antibody (1:200 rabbit anti-human MRP2

192

antibody) at 4oC overnight. After two washes with PBS buffer saline Tween-20

193

(PBST), the membrane was washed with PBS buffer and incubated with secondary

194

antibody for 2h at room temperature. Immunopositive bands were detected using

195

enhanced chemiluminescent western blotting reagents (Pierce, Nashville, TN, USA).

196

Levels of GAPDH were used as internal control to account for total loading.

197

Experiments were conducted three times.

198

Statistical analysis

199

For all assays, 3 independent trials were performed, and each trial was replicated

200

6 times. Results were expressed as mean ± standard errors. Statistical analyses

201

between MWCNTs treated and control samples were calculated using one-way

202

analysis of variance (ANOVA) with Tukey post hoc test by Graphpad Prism 6.

203

Statistical significance was set at p < 0.05.

RESULTS

204 205

Characteristics of MWCNTs

206

Structural parameters of the six MWCNTs from Nanjing XFNANO Materials

207

Tech Co Ltd. are shown in Table 1. The length of long MWCNTs ranges from 10 µm

208

to 30µm, which are about 20-times longer than short MWCNTs (0.5-2µm). The

209

contents of hydroxyl and carboxyl groups in functionalized MWCNTs were 5.58% 9



210

and 3.86%, respectively. TEM was used to characterize the six MWCNTs. As shown

211

in Figure S1, the six MWCNTs were “bamboo like”. Determination of the outside

212

diameter was similar to what has been reported by the manufacturer. Further, FTIR

213

spectra of functionalized MWCNTs analyses found that the peak at approximate

214

1714cm-1 can be attributed to the stretching vibration of carbon-oxygen group, while

215

it was negligible in the pristine MWCNTs (Figure S2).

216

Effect of MWCNTs on HepG2 cell viability

217

The 24h exposure of six MWCNTs significantly decreased the cell viability of

218

HepG2 and showed a concentration-depend response (Figure 1). M-L-COOH and

219

M-L-OH at 2µg/mL and higher concentrations caused the significant decreases in cell

220

viability. However, the lowest effective concentrations for M-L, M-S-COOH, and

221

M-S were 5µg/mL, and for M-S-OH it was 10µg/mL. With the increase of exposure

222

concentration, M-L-COOH induced lower cell viability than M-L-OH and M-L,

223

indicating that functionalization of long MWCNTs increased the cytotoxicity.

224

However, the functionalization of short MWCNTs decreased their cytotoxicity.

225

Further, for MWCNTs with the same functional groups, M-L-COOH induced higher

226

cytotoxicity than M-S-COOH, but the M-L caused lower cytotoxicity than M-S

227

(Figures S3).

228

Generation of intracellular ROS

229

Exposure of the six MWCNTs significantly increased the intracellular ROS

230

generation, which was based on the increase of DCF fluorescence values (Figures 2A

231

and 2B). The long MWCNTs with or without functionalized groups significantly

232

increased ROS generation at 2µg/mL and higher concentrations. However the lowest

233

effective concentration for the three short MWCNTs was 5µg/mL. With increased of

234

exposure concentration, M-L-COOH and M-L-OH induced more ROS generation 10


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than M-L. However, M-S induced more ROS generation than M-S-COOH and

236

M-S-OH. Further, M-S induced more ROS generation than M-L. However, when the

237

MWCNTs were functionalized by the hydroxyl and carboxyl groups, M-L-COOH and

238

M-L-OH caused more ROS generation than M-S-COOH and M-S-OH (Figures S4).

239

Changes in mitochondrial membrane potential

240

Mitochondrial membrane potential was analyzed using the JC1 assay, in which

241

mitochondrial depolarization (early indicator of cell apoptosis) was determined by

242

decrease of red/green fluorescence intensity ratio23. The results showed that the six

243

MWCNTs significantly decreased the red/green ratio of JC1 at 2µg/mL and higher

244

concentrations (Figures 2C and 2D), indicating the mitochondrial depolarization and

245

potential cell apoptosis. The different functionalization in long and short MWCNTs

246

showed that carboxyl functionalized MWCNTs and pristine MWCNTs caused lower

247

red/green ratio of JC1 compared to hydroxyl functionalized MWCNTs, but no

248

significant differences were found between the long and short MWCNTs with the

249

same functional groups (Figure S5). For pristine MWNCTs, the M-L caused lower

250

lower red/green ratio of JC1 than M-S (p M-S-COOH = M-S = M-L >

273

M-L-OH > M-L-COOH. CAM accumulation in HepG2 was also verified by

274

microscope images (Figure S8).

275

Influence of MWCNTs on arsenic toxicity

276

The As was chosen as the target pollutant to identify the chemosensitizing

277

activity of MWCNTs. As within the cell can be conjugated by glutathione (GSH) into

278

As-GSH, which is a substrate of ABCCs/MRPs24. ROS generation, one of the

279

mechanisms of As toxicity was chosen as the toxic endpoint. Two strategies of

280

co-exposure were applied. Results showed that MK571 significantly increased ROS

281

generation caused by As alone at non-toxic concentration with the both strategies

282

(Figure 4), indicating the inhibition of ABC transporter activities indeed increased As

283

toxicity. For co-exposure of As and MWCNTs, MWCNTs at non-toxic concentrations

284

also significantly increased the ROS generation induced by As with the both strategies 12


Page 12 of 34

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285

used in this study. For long MWCNTs, functional groups significantly increased their

286

chemosensitizing activity and –OH group had greater influence than –COOH group.

287

However, for short MWCNTs, the functional groups decreased the chemosensitizing

288

activity of MWCNTs, and –COOH group had greater influence than –OH group.

289

Cell membrane damage

290

Membrane fluidity plays an important role in membrane functions, including the

291

functions of membrane ABC transporters. In this study, membrane fluidity of HepG2

292

was measured by detecting fluorescence polarization of the TMA-DPH. Polarization

293

ratio (P) values of TMA-DPH in HepG2 exposed to the six MWCNTs are shown in

294

Figure 5. Exposure of MWCNTs significantly decreased the P values, indicating an

295

increased membrane fluidity. Similar results have been found in the impact of carbon

296

nanotubes on membrane fluidity of bacteria25. Further, the lowest effective

297

concentrations in membrane fluidity were very similar with the lowest effective

298

concentrations of CAM accumulation, indicating changes in membrane fluidity might

299

be the potential reason for inhibition of ABC transporter activity.

300

Expression of MRP2 in HepG2

301

Western blotting was used to measure the expression of MRP2 in HepG2.

302

Results showed that the MWCNTs at low concentration did not significantly change

303

the expression of MRP2 protein, except for the M-S-OH and M-L (Figure S10). No

304

decrease in expression that would correlate with decreased transporter activity was

305

observed.

306

307

DISCUSSION

308

In the present study we assessed the toxic effects of six MWCNTs with different

309

lengths and functional groups. To date there are very few studies that simultaneously 13



310

compare toxic effects of different MWCNTs in a single study. Further, the

311

chemosensitizing effects of low levels of MWCNTs have previously not been

312

considered. Our results show that MWCNTs with different lengths and functional

313

groups can reduce cell viability, and concentration-dependent relationships were

314

identified. Increased ROS generation has been shown to be one of the primary

315

mechanisms by which MWCNTs induce cytotoxicity26, 27. Many studies have found

316

that MWCNTs could induce ROS generation and oxidative stress28-30. Similar to these

317

studies, our results showed that the six MWCNTs investigated induced ROS

318

generation, and the effective concentrations on ROS generation were very similar to

319

those found to impact cell viability (Figures 1 and 2). Additionally, many studies have

320

shown that ROS generation is linked to mitochondrial oxidative damage and

321

apoptosis. Our results found that MWCNTs exposure induced mitochondrial

322

depolarization, indicating potential cell apoptosis (Figure 2). However, since the ROS

323

generation and changes in mitochondrial membrane potential were influenced by

324

different pathways, differences in potency in ROS and mitochondrial membrane

325

potentials were found among the six MWCNTs. Further, we found an increase in

326

lysosomes after following MWCNTs exposure, which indirectly indicates that cells

327

uptake of MWCNTs. The nanotubes entering the cells were found to have ability

328

inducing oxidative stress by disturbing the balance between oxidant and antioxidant

329

processes, e.g. the glutathione system31. Thus, the increased ROS generation in

330

HepG2 cells exposed to MWCNTs might be due to the cell uptake of MWCNTs.

331

According to the above results, the lowest effective concentrations for six MWCNTs

332

on cell viability, ROS generation, lysosome and mitochondrial membrane potential

333

were very similar. Thus, we can deduce that when MWCNTs are exposed to HepG2

14


Page 14 of 34

Page 15 of 34


334

cells, they enter into the cell, then increase ROS generation and damage cell function

335

(mitochondrial membrane potential) to induce cytotoxicity (decrease of cell viability).

336

The MWCNTs with different lengths and functional groups induced different

337

degree of cytotoxicity. Table 2 summarized the toxicity of different MWCNTs. M-S

338

induced higher cytotoxicity than M-L, which might be due to the possibility that the

339

shorter ones could more easily enter into the cells32. For example, Monteiro-Riviere et

340

al.33 showed that MWCNTs could enter into human keratinocytes, however the longer

341

nanotubes tended to form aggregates, thus decreasing their toxicity. Han et al.34 also

342

found that the smaller sized MWCNTs were more toxic than the longer ones, possibly

343

because the smaller nanotubes were easier to be incorporated into the cytoplasm. We

344

found that when MWCNTs were functionalized, M-L-COOH and M-L-OH induced a

345

greater toxicity than M-L. Similar results were found in the study from Patlolla et al.35,

346

which found that long functionalized MWCNTs (lengths of 15-20 µm) had a higher

347

clastogenic and genotoxic potential compared to non-functionalized form. The

348

functionalization with –COOH and -OH might be a potential way to improve

349

solubility of MWCNTs in water, which may make the actual exposure concentration

350

higher via increased bioavailability36, increasing cell-nanotube contact10, thus leading

351

to the higher cytotoxicity. However, for short MWCNTs, M-S-COOH and M-S-OH

352

induced lower toxicity than M-S, which might be due to the functionalization with –

353

COOH and -OH further improve the solubility and biocompatibility of short

354

MWCNTs37, which results in lower toxicity38. Based on above results,

355

functionalization could increase the solubility of long MWCNTs and reduce their

356

aggregation, resulting in an increase of cytotoxicity. However, functionalization

357

allows short MWCNTs to easily enter into cells and improves their biocompatibility,

358

resulting in a decrease in cytotoxicity. 15



359

For low concentrations (

Comparison of Cytotoxicity and Inhibition of Membrane ABC

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